Method of analysing a blood sample of a subject for the presence of a disease marker

ABSTRACT

The present invention relates to a method of analysing a blood sample of a subject for the presence of a disease marker, said method comprising the steps of a) extracting nucleic acid from anucleated blood cells in said blood sample to provide an anucleated blood cells-extracted nucleic acid fraction, and b) analysing said anucleated blood cells-extracted nucleic acid fraction for the presence of a disease marker, wherein said disease marker is a disease-specific mutation in a gene of a cell of said subject, or wherein said disease marker is a disease-specific expression profile of genes of a cell of said subject.

RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.14/006,089, which is the U.S. National Stage of InternationalApplication No. PCT/NL2012/050025, filed Jan. 16, 2012, published inEnglish, and claims the benefit of European Application No. 11158912.3,filed on Mar. 18, 2011, European Application No. 11167973.4, filed onMay 27, 2011, and International Application No. PCT/NL2011/050518, filedon Jul. 15, 2011, the entire teachings of the above applications areincorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Nov. 25, 2013, isnamed P91312US10 sequence list_ST25.txt and is 8 KB in size.

FIELD OF THE INVENTION

The invention is in the field of medical diagnostics, in particular inthe field of disease diagnostics and monitoring. The invention isdirected to markers for the detection of disease, to methods fordetecting disease, and to a method for determining the efficacy of adisease treatment.

BACKGROUND OF THE INVENTION

In clinical practice there is a strong need to be able to detect diseasein its earliest stages, to predict disease progression, and to implementpatient-tailored therapy. Early detection of in particular neoplasticdisease (cancer) is critical to ensure favourable treatment of thedisease. In spite of numerous advances in medical research, cancerremains a major cause of death worldwide. When patients seek treatment,they are generally exhibiting symptoms of distant metastases, meaningthat too often the cancer is detected too late.

Lung, prostate, breast, and colon cancer are the most common tumours,and in order to facilitate appropriate remedial action by surgicalresection, radiotherapy, chemotherapy, or other known treatment methodsthere is a need for rapid and simple methods for the early diagnoses ofcancer. The availability of good diagnostic methods for cancer is alsoimportant to assess patient responses to treatment, or to assessrecurrence due to re-growth at the original site or metastasis.

Several types of cancer markers, such as, for example, oncogeneproducts, growth factors and growth factor receptors, angiogenicfactors, proteases, adhesion factors and tumour suppressor geneproducts, etc, are presently known and are not only considered essentialfor early diagnosis, but also for differential diagnosis of patientswith uncertain clinical abnormalities such as for distinguishingmalignant from benign abnormalities; for predicting the likelihood ofresponse in a particular patient with established malignancy to aselected therapeutic method of treatment; and for providing informationconcerning the risk, presence, status, or future behaviour of themalignancy in a human or animal subject. Currently, the ability todetect and diagnose cancer through the detection of tumour or cancermarkers is an area of widespread interest and as a consequence the needexists for reproducible and reliable methods of identifying new and moreuseful cancer markers in patient specimens.

Glioblastoma is the most common and most aggressive type of primarybrain tumor in humans. The disease is difficult to diagnose and evenharder to treat due, in part, to the blood-brain barrier that hindersthe delivery of therapeutic agents and detection of potentiallyimportant diagnostic markers. Diagnostic markers for glioblastoma areavailable, but are specific for the tumour tissue itself and require atumour sample.

Improved screening and detection methods are needed in order to detectcancer in an early phase and to follow the progression of the disease.In the case of cancer we are at a state where we do not only need todetect the tumour, but also need to detect it before it has reached apoint of no return, where the treatment becomes palliative instead ofcurative. People at risk, as well as patients with recurring cancer,should be monitored extensively. Furthermore, since tumours can responddifferently to different therapeutics, patient stratification isbecoming of importance.

Genetic analysis using tumour biopsies has allowed the identification ofmany mutations that are useful for diagnosis of the cancer as well asfor emerging patient stratification strategies. However, a disadvantageof current genetic analysis of tumours is the need for tumour biopsies,which are often impossible to dissect from patients. Furthermore, theuse of biopsies is static and does not allow genetic monitoring oftumour progression or recurrence over time. Moreover, many tumours areheterogeneous, resulting in potential false-positive or false-negativegenetic characterization of biopsies of such tumours.

Recently, the use of circulating tumour cells for diagnosis andmonitoring of tumour progression or recurrence showed the use of bloodas a source of tumour-derived material, notably tissue fragments in theform of cells. However, the use of circulating tumour cells isinefficient for most cancers.

Calverley et al (Clinical and Translational Science vol 3, issue 5,2010) disclose a down-regulation of gene expression of platelets inmetastatic lung cancer. The authors identified 200 genes that appearedto be differentially expressed between healthy persons and patients.According to the authors, the platelet proteome is mirrored in thetranscriptome of the platelet. The gene expression as measured wascorrelated to genes from megakaryocytes. No disclosure is made thatRNA/DNA derived from other cells than megakaryocytes was measured whentesting the thrombocytes, and no indication that circulating RNA/DNAderived from other cells can be taken up by thrombocytes.

In general, a disease marker is defined as a compound of which theconcentration is altered, preferably elevated, in a biological fluidfrom a diseased patient when compared to a normal healthy subject, andwhich may subsequently be used as a marker compound indicative of adisease. Yet, the identification of specific compounds, for instanceproteins, in various body fluids as markers of disease, such as cancer,has been hampered by the lack of suitable techniques therefore.

Also in case of diseases other than cancer, markers may be availablethat are difficult to detect. This hampers early diagnosis of thedisease.

Lood et al (Blood vol 116, no 11, 2010) disclose that the geneexpression of IFN-I regulated genes in platelets in patients with SLE isincreased. The authors hypothesize that IFNalpha influences geneexpression in megakaryocytes, resulting in an increased level of IFN-Iregulated proteins in platelets. The expressions of genes frommegakaryocytes are thus correlated to SLE or vascular disease. Nodisclosure is made that RNA/DNA from diseased cells may be taken up byplatelets.

The present invention aims to overcome the problem of the prior art thatnot all diseased tissues or disease types (e.g. tumours) result incirculating disease cells (e.g. circulating tumour cells). The presentinvention also aims to overcome the problem that protein markers fordetecting diseases such as cancer are difficult to detect. Further, thepresent invention aims to provide methods that do not require biopsies,and allow extensive monitoring of patients.

SUMMARY OF THE INVENTION

The present invention in a first aspect provides a method of analysing ablood sample of a subject for the presence of a disease marker, saidmethod comprising the steps of a) extracting nucleic acid fromanucleated blood cells, preferably thrombocytes, in said blood sample toprovide an anucleated blood cell-extracted nucleic acid fraction, and b)analysing said anucleated blood cell-extracted nucleic acid fraction forthe presence of a disease marker, wherein said disease marker is adisease-specific mutation in a gene of a nucleated cell of said subject,or wherein said disease marker is a disease-specific expression profileof genes of a nucleated cell of said subject.

It was surprisingly found that nucleic acids from nucleated cells arepresent in anucleated blood cells such as thrombocytes. It may be thatnucleated cells excrete nucleic acids into the blood stream and theseexcreted nucleic acids are then taken up from the blood stream byanucleated cells such as thrombocytes or that in some other way oftransport nucleic acids from nucleated cells are transferred toanucleated blood cells. The inventors realized for the first time thatdisease markers may be used on the nucleic acids extracted fromanucleated blood cells to identify diseases from nucleated cells.

In a preferred embodiment of the method of the invention said anucleatedblood cell-extracted nucleic acid fraction comprises nucleic acidoriginating from a nucleated cell. In a preferred embodiment of thepresent invention and embodiments thereof, the anucleated bloodcell-extracted nucleic acid fraction is not megakaryocyte-derivednucleic acid or megakaryocyte-derived RNA, i.e. the nucleic acidfraction to be tested is not of megakaryocyte-lineage or megakaryocytegenomic origin.

The term “anucleated blood cell” as used herein refers to a cell thatlacks a nucleus. The term includes reference to both erythrocyte andthrombocyte. Preferred embodiments of anucleated cells in aspects ofthis invention are thrombocytes. The term “anucleated blood cell”preferably does not include reference to cells that lack a nucleus as aresult of faulty cell division.

The term “nucleated cell” as used herein refers to a cell having anucleus. The term includes reference to somatic cells, germ cells andstem cells, and may include cells from colon, pancreas, brain, bladder,breast, prostate, lung, breast, ovary, uterus, liver, kidney, spleen,thymus, thyroid, nerve tissue, connective tissue, blood, epithelialtissue, lymph node, bone, muscle and skin tissues. The nucleated cell ispreferably a cell from a diseased tissue. In a preferred embodiment, thenucleated cell is not a megakaryocyte.

Thus, the present invention is generally aimed at analysing nucleicacids that have been transferred from cells that have a nucleus intocells that have no nucleus, wherein the cells that have no nucleus canbe easily isolated from the blood stream and contain nucleic acid fromthe nucleated cells.

The term “nucleus” refers to the membrane-enclosed organelle found ineukaryotic cells that contains most of the cell's genetic materialorganized in the form of chromosomes. The genes within these chromosomesare the cell's nuclear genome. The interior of the nucleus contains anumber of subnuclear bodies including the RNA-comprising nucleolus,which is mainly involved in the assembly of RNA-comprising ribosomes.After being produced in the nucleolus, ribosomes are exported to thecytoplasm where they translate mRNA.

An anucleated blood cell-extracted nucleic acid fraction preferablyrefers to a fraction comprising chromosomal DNA, ribosomal RNA,nucleolus RNA, and/or messenger RNA.

The term “gene” as used herein, and in particular in the phrasing“mutation in a gene of a nucleated cell” is meant to refer to anynucleic acid sequence, both chromosomal and extra-chromosomal, of anucleated (somatic) cell, preferably a nuclear nucleic acid sequence,and may include transcribed and non-transcribed sequences as well asribosomal RNA sequences, most preferably chromosomal sequences that aretranscribed into RNA.

In a preferred embodiment of a method of the invention saiddisease-specific mutation is in a chromosomal gene.

In another preferred embodiment, said gene is not a gene from ananucleated blood cell. In another preferred embodiment, said gene is nota gene from a megakaryocyte. In yet another preferred embodiment saidgene is not CD109.

In a preferred embodiment of a method of the invention saiddisease-specific expression profile is the expression profile ofchromosomal genes. In particular of chromosomal genes from a nucleatedcell the mRNA of which is present in a thrombocyte.

In another preferred embodiment of a method of the invention saidnucleic acid is ribonucleic acid (RNA), more preferably messengerribonucleic acid (mRNA).

In a preferred embodiment of a method of the invention said nucleic acidis not mtDNA. Hence, mitochondrial nucleic acid is preferably not anaspect of the present invention.

In another preferred embodiment of a method of analysing a blood sampleaccording to the invention said step b) of analysing said anucleatedblood cell-extracted nucleic acid fraction for the presence of a diseasemarker comprises the selective amplification of:

i) said mutation by reverse transcriptase polymerase chain reactionamplification using at least one nucleic acid mutation-specificamplification primer or probe, orii) a plurality of mRNAs by reverse transcriptase polymerase chainreaction amplification to determine the expression level of thechromosomal genes encoding said mRNAs to thereby provide an expressionprofile for said genes and comparing said expression profile to areference profile.

The blood sample is preferably outside the body.

In a preferred embodiment of a method of the invention the disease isselected from the group consisting of cancer, autoimmune disease, skindiseases, eye disease, endocrine diseases, neurological disorders, andcardiovascular diseases.

In another preferred embodiment of a method of the invention saiddisease is selected from the group consisting of autoimmune disease,skin diseases, eye disease, endocrine diseases, neurological disorders,and cardiovascular diseases.

In another preferred embodiment of a method of the invention saiddisease is cancer.

In yet another preferred embodiment of a method of the invention saidcancer is a solid tumour cancer, preferably selected from colon,pancreas, brain, bladder, breast, prostate, lung, breast, ovary, uterus,liver, kidney, spleen, thymus, thyroid, nerve tissue, epithelial tissue,lymph node, bone, muscle and skin.

In another preferred embodiment of a method of the invention saiddisease is not cancer.

In another preferred embodiment of a method of the invention saiddisease is not a vascular disease.

In another preferred embodiment of a method of the invention saiddisease is not systemic lupus erythematosus.

In another preferred embodiment of a method of the invention saiddisease is not sickle cell disease.

In another preferred embodiment of a method of the invention saiddisease is not Alzheimer's disease.

In another preferred embodiment of a method of the invention saiddisease is not a disease associated with pathological megakaryocytefunction.

In another preferred embodiment of a method of the invention saiddisease is not a disease associated with pathological platelet function.

The above embodiments that are disclaimed in preferred embodiments maybe combined in any combination.

In another preferred embodiment of a method of the invention saiddisease is selected from the group consisting of autoimmune disease,skin diseases, eye disease, endocrine diseases, and neurologicaldisorders.

In preferred embodiments of aspects of the invention the auto-immunedisease is selected from the group consisting of Achlorhydra AutoimmuneActive Chronic Hepatitis; Acute Disseminated Encephalomyelitis; Acutehemorrhagic leukoencephalitis; Addison's Disease; Agammaglobulinemia;Alopecia areata; Amyotrophic Lateral Sclerosis; Ankylosing Spondylitis;Anti-GBM/TBM Nephritis; Antiphospholipid syndrome; Antisynthetasesyndrome; polyarticular Arthritis; Atopic allergy; Atopic Dermatitis;Autoimmune Aplastic Anemia; Autoimmune cardiomyopathy; Autoimmuneenteropathy; Autoimmune hemolytic anemia; Autoimmune hepatitis;Autoimmune inner ear disease; Autoimmune lymphoproliferative syndrome;Autoimmune peripheral neuropathy; Autoimmune pancreatitis; Autoimmunepolyendocrine syndrome; Autoimmune progesterone dermatitis; Autoimmunethrombocytopenic purpura; Autoimmune uveitis; Balo disease/Baloconcentric sclerosis; Bechets Syndrome; Berger's disease; Bickerstaffsencephalitis; Blau syndrome; Bullous Pemphigoid; Castleman's disease;Celiac disease; Chagas disease; Chronic Fatigue Immune DysfunctionSyndrome; Chronic inflammatory demyelinating polyneuropathy; Chronicrecurrent multifocal osteomyelitis; Chronic lyme disease; Chronicobstructive pulmonary disease; Churg-Strauss syndrome; CicatricialPemphigoid; Coeliac Disease; Cogan syndrome; Cold agglutinin disease;Complement component 2 deficiency; Cranial arteritis; CREST syndrome;Crohns Disease; Cushing's Syndrome; Cutaneous leukocytoclastic angiitis;Dego's disease; Dercum's disease; Dermatitis herpetiformis;Dermatomyositis; Diabetes mellitus type 1; Diffuse cutaneous systemicsclerosis; Dressler's syndrome; Discoid lupus erythematosus; Eczema;Endometriosis; Enthesitis-related arthritis; Eosinophilic fasciitis;Eosinophilic gastroenteritis; Epidermolysis bullosa acquisita; Erythemanodosum; Essential mixed cryoglobulinemia; Evan's syndrome;Fibrodysplasia ossificans progressiva; Fibromyalgia/Fibromyositis;Fibrosing aveolitis; Gastritis; Gastrointestinal pemphigoid; Giant cellarteritis; Glomerulonephritis; Goodpasture's syndrome; Graves' disease;Guillain-Barre syndrome; Hashimoto's encephalitis; Hashimoto'sthyroiditis; Haemolytic anaemia; Henoch-Schonlein purpura; Herpesgestationis; Hidradenitis suppurativa; Hughes syndrome;Hypogammaglobulinemia; Idiopathic Inflammatory Demyelinating Diseases;Idiopathic pulmonary fibrosis; Idiopathic thrombocytopenic purpura; IgAnephropathy; Inclusion body myositis; Inflammatory demyelinating polyneuop athy; Interstitial cystitis; Irritable Bowel Syndrome (IBS);Juvenile idiopathic arthritis; Juvenile rheumatoid arthritis; Kawasaki'sDisease; Lambert-Eaton myasthenic syndrome; Leukocytoclastic vasculitis;Lichen planus; Lichen sclerosus; Linear IgA disease; Lou Gehrig'sDisease; Lupoid hepatitis; Lupus erythematosus; Majeed syndrome;Ménière's disease; Microscopic polyangiitis; Miller-Fisher syndrome;Mixed Connective Tissue Disease; Morphea; Mucha-Habermann disease;Muckle-Wells syndrome; Multiple Myeloma; Multiple Sclerosis; Myastheniagravis; Myositis; Narcolepsy; Neuromyelitis optica; Neuromyotonia;Occular cicatricial pemphigoid; Opsoclonus myoclonus syndrome; Ordthyroiditis; Palindromic rheumatism; PANDAS; Paraneoplastic cerebellardegeneration; Paroxysmal nocturnal hemoglobinuria; Parry Rombergsyndrome; Parsonnage-Turner syndrome; Pars planitis; Pemphigus;Pemphigus vulgaris; Pernicious anaemia; Perivenous encephalomyelitis;POEMS syndrome; Polyarteritis nodosa; Polymyalgia rheumatica;Polymyositis; Primary biliary cirrhosis; Primary sclerosing cholangitis;Progressive inflammatory neuropathy; Psoriasis; Psoriatic Arthritis;Pyoderma gangrenosum; Pure red cell aplasia; Rasmussen's encephalitis;Raynaud phenomenon; Relapsing polychondritis; Reiter's syndrome;Restless leg syndrome; Retroperitoneal fibrosis; Rheumatoid arthritis;Rheumatoid fever; Sarcoidosis; Schizophrenia; Schmidt syndrome;Schnitzler syndrome; Scleritis; Scleroderma; Sjögren's syndrome;Spondyloarthropathy; Sticky blood syndrome; Still's Disease; Stiffperson syndrome; Subacute bacterial endocarditis (SBE); Susac'ssyndrome; Sweet syndrome; Sydenham Chorea; Sympathetic ophthalmia;Takayasu's arteritis; Temporal arteritis; Tolosa-Hunt syndrome;Transverse Myelitis; Ulcerative Colitis; Undifferentiated connectivetissue disease; Undifferentiated spondyloarthropathy; Vasculitis;Vitiligo; Wegener's granulomatosis; Wilson's syndrome; andWiskott-Aldrich syndrome.

In other preferred embodiments of aspects of the invention the skindisease is selected from the group consisting of Acneiform eruptions;Autoinflammatory syndromes; Chronic blistering; Conditions of the mucousmembranes; Conditions of the skin appendages; Conditions of thesubcutaneous fat; Congenital anomalies; Connective tissue diseases (suchas Abnormalities of dermal fibrous and elastic tissue); Dermal andsubcutaneous growths; Dermatitis (including Atopic Dermatitis, ContactDermatitis, Eczema, Pustular Dermatitis, and Seborrheic Dermatitis);Disturbances of pigmentation; Drug eruptions; Endocrine-related skindisease; Eosinophilic; Epidermal nevi, neoplasms, cysts; Erythemas;Genodermatoses; Infection-related skin disease; Lichenoid eruptions;Lymphoid-related skin disease; Melanocytic nevi and neoplasms (includingMelanoma); Monocyte- and macrophage-related skin disease; Mucinoses;Neurocutaneous; Noninfectious immunodeficiency-related skin disease;Nutrition-related skin disease; Papulosquamous hyperkeratotic (includingPalmoplantar keratodermas); Pregnancy-related skin disease; Pruritic;Psoriasis; Reactive neutrophilic; Recalcitrant palmoplantar eruptions;Resulting from errors in metabolism; Resulting from physical factors(including Ionizing radiation-induced); Urticaria and angioedema;Vascular-related skin disease.

In other preferred embodiments of aspects of the invention the endocrinedisease is selected from the group consisting of Adrenal disorders;Glucose homeostasis disorders; Thyroid disorders; Calcium homeostasisdisorders and Metabolic bone disease; Pituitary gland disorders; and Sexhormone disorders.

In other preferred embodiments of aspects of the invention the eyedisease is selected from the group consisting of H00-H06 Disorders ofeyelid, lacrimal system and orbit; H10-H13 Disorders of conjunctiva;H15-H22 Disorders of sclera, cornea, iris and ciliary body; H25-H28Disorders of lens; H30-H36 Disorders of choroid and retina (includingH30 Chorioretinal inflammation, H31 Other disorders of choroid, H32Chorioretinal disorders in diseases classified elsewhere, H33 Retinaldetachments and breaks, H34 Retinal vascular occlusions, H35 Otherretinal disorders, and H36 Retinal disorders in diseases classifiedelsewhere); H40-H42 Glaucoma; H43-H45 Disorders of vitreous body andglobe; H46-H48 Disorders of optic nerve and visual pathways; H49-H52Disorders of ocular muscles, binocular movement, accommodation andrefraction; H53-H54.9 Visual disturbances and blindness; and H55-H59Other disorders of eye and adnexa.

In other preferred embodiments of aspects of the invention theneurological disorder is selected from the group consisting ofAbarognosis; Acquired Epileptiform Aphasia; Acute disseminatedencephalomyelitis; Adrenoleukodystrophy; Agenesis of the corpuscallosum; Agnosia; Aicardi syndrome; Alexander disease; Alien handsyndrome; Allochiria; Alpers' disease; Alternating hemiplegia;Alzheimer's disease; Amyotrophic lateral sclerosis (see Motor NeuroneDisease); Anencephaly; Angelman syndrome; Angiomatosis; Anoxia; Aphasia;Apraxia; Arachnoid cysts; Arachnoiditis; Arnold-Chiari malformation;Arteriovenous malformation; Ataxia Telangiectasia; Attention deficithyperactivity disorder; Auditory processing disorder; AutonomicDysfunction; Back Pain; Batten disease; Behcet's disease; Bell's palsy;Benign Essential Blepharospasm; Benign Intracranial Hypertension;Bilateral frontoparietal polymicrogyria; Binswanger's disease;Blepharospasm; Bloch-Sulzberger syndrome; Brachial plexus injury; Brainabscess; Brain damage; Brain injury; Brain tumor; Brown-Séquardsyndrome; Canavan disease; Carpal tunnel syndrome; Causalgia; Centralpain syndrome; Central pontine myelinolysis; Centronuclear myopathy;Cephalic disorder; Cerebral aneurysm; Cerebral arteriosclerosis;Cerebral atrophy; Cerebral gigantism; Cerebral palsy; Cerebralvasculitis; Cervical spinal stenosis; Charcot-Marie-Tooth disease;Chiari malformation; Chorea; Chronic fatigue syndrome; Chronicinflammatory demyelinating polyneuropathy (CIDP); Chronic pain; CoffinLowry syndrome; Coma; Complex regional pain syndrome; Compressionneuropathy; Congenital facial diplegia; Corticobasal degeneration;Cranial arteritis; Craniosynostosis; Creutzfeldt-Jakob disease;Cumulative trauma disorders; Cushing's syndrome; Cytomegalic inclusionbody disease (CIBD); Cytomegalovirus Infection; Dandy-Walker syndrome;Dawson disease; De Morsier's syndrome; Dejerine-Klumpke palsy;Dejerine-Sottas disease; Delayed sleep phase syndrome; Dementia;Dermatomyositis; Developmental dyspraxia; Diabetic neuropathy; Diffusesclerosis; Dravet syndrome; Dysautonomia; Dyscalculia; Dysgraphia;Dyslexia; Dystonia; Empty sella syndrome; Encephalitis; Encephalocele;Encephalotrigeminal angiomatosis; Encopresis; Epilepsy; Erb's palsy;Erythromelalgia; Essential tremor; Fabry's disease; Fahr's syndrome;Fainting; Familial spastic paralysis; Febrile seizures; Fisher syndrome;Friedreich's ataxia; Fibromyalgia; Gaucher's disease; Gerstmann'ssyndrome; Giant cell arteritis; Giant cell inclusion disease; GloboidCell Leukodystrophy; Gray matter heterotopia; Guillain-Barré syndrome;HTLV-1 associated myelopathy; Hallervorden-Spatz disease; Head injury;Headache; Hemifacial Spasm; Hereditary Spastic Paraplegia; Heredopathiaatactica polyneuritiformis; Herpes zoster oticus; Herpes zoster;Hirayama syndrome; Holoprosencephaly; Huntington's disease;Hydranencephaly; Hydrocephalus; Hypercortisolism; Hypoxia;Immune-Mediated encephalomyelitis; Inclusion body myositis;Incontinentia pigmenti; Infantile phytanic acid storage disease;Infantile Refsum disease; Infantile spasms; Inflammatory myopathy;Intracranial cyst; Intracranial hypertension; Joubert syndrome; Karaksyndrome; Kearns-Sayre syndrome; Kennedy disease; Kinsbourne syndrome;Klippel Feil syndrome; Krabbe disease; Kugelberg-Welander disease; Kuru;Lafora disease; Lambert-Eaton myasthenic syndrome; Landau-Kleffnersyndrome; Lateral medullary (Wallenberg) syndrome; Learningdisabilities; Leigh's disease; Lennox-Gastaut syndrome; Lesch-Nyhansyndrome; Leukodystrophy; Lewy body dementia; Lissencephaly; Locked-Insyndrome; Lou Gehrig's disease (See Motor Neurone Disease); Lumbar discdisease; Lumbar spinal stenosis; Lyme disease—Neurological Sequelae;Machado-Joseph disease (Spinocerebellar ataxia type 3); Macrencephaly;Macropsia; Megalencephaly; Melkersson-Rosenthal syndrome; Menieresdisease; Meningitis; Menkes disease; Metachromatic leukodystrop hy;Microcephaly; Micropsia; Migraine; Miller Fisher syndrome; Mini-stroke(transient ischemic attack); Mitochondrial myopathy; Mobius syndrome;Monomelic amyotrophy; Motor Neurone Disease; Motor skills disorder;Moyamoya disease; Mucopolysaccharidoses; Multi-infarct dementia;Multifocal motor neuropathy; Multiple sclerosis; Multiple systematrophy; Muscular dystrophy; Myalgic encephalomyelitis; Myastheniagravis; Myelinoclastic diffuse sclerosis; Myoclonic Encephalopathy ofinfants; Myoclonus; Myopathy; Myotubular myopathy; Myotonia congenita;Narcolepsy; Neurofibromatosis; Neuroleptic malignant syndrome;Neurological manifestations of AIDS; Neurological sequelae of lupus;Neuromyotonia; Neuronal ceroid lip ofuscinosis; Neuronal migrationdisorders; Niemann-Pick disease; Non 24-hour sleep-wake syndrome;Nonverbal learning disorder; O'Sullivan-McLeod syndrome; OccipitalNeuralgia; Occult Spinal Dysraphism Sequence; Ohtahara syndrome;Olivopontocerebellar atrophy; Opsoclonus myoclonus syndrome; Opticneuritis; Orthostatic Hypotension; Overuse syndrome; Palinopsia;Paresthesia; Parkinson's disease; Paramyotonia Congenita; Paraneoplasticdiseases; Paroxysmal attacks; Parry-Romberg syndrome;Pelizaeus-Merzbacher disease; Periodic Paralyses; Peripheral neuropathy;Persistent Vegetative State; Pervasive developmental disorders; Photicsneeze reflex; Phytanic acid storage disease; Pick's disease; Pinchednerve; Pituitary tumors; PMG; Polio; Polymicrogyria; Polymyositis;Porencephaly; Post-Polio syndrome; Postherpetic Neuralgia (PHN);Postinfectious Encephalomyelitis; Postural Hypotension; Prader-Willisyndrome; Primary Lateral Sclerosis; Prion diseases; Progressivehemifacial atrophy; Progressive multifocal leukoencephalopathy;Progressive Supranuclear Palsy; Pseudotumor cerebri; Rabies; Ramsay-Huntsyndrome (Type I and Type II); Rasmussen's encephalitis; Reflexneurovascular dystrophy; Refsum disease; Repetitive motion disorders;Repetitive stress injury; Restless legs syndrome; Retrovirus-associatedmyelopathy; Rett syndrome; Reye's syndrome; Rhythmic Movement Disorder;Romberg syndrome; Saint Vitus dance; Sandhoff disease; Schizophrenia;Schilder's disease; Schizencephaly; Sensory integration dysfunction;Septo-optic dysplasia; Shaken baby syndrome; Shingles; Shy-Dragersyndrome; Sjögren's syndrome; Sleep apnea; Sleeping sickness;Snatiation; Sotos syndrome; Spasticity; Spina bifida; Spinal cordinjury; Spinal cord tumors; Spinal muscular atrophy; Spinocerebellarataxia; Steele-Richardson-Olszewski syndrome; Stiff-person syndrome;Stroke; Sturge-Weber syndrome; Subacute sclerosing panencephalitis;Subcortical arteriosclerotic encephalopathy; Superficial siderosis;Sydenham's chorea; Syncope; Synesthesia; Syringomyelia; Tarsal tunnelsyndrome; Tardive dyskinesia; Tarlov cyst; Tay-Sachs disease; Temporalarteritis; Tetanus; Tethered spinal cord syndrome; Thomsen disease;Thoracic outlet syndrome; Tic Douloureux; Todd's paralysis; Tourettesyndrome; Toxic encephalopathy; Transient ischemic attack; Transmissiblespongiform encephalopathies; Transverse myelitis; Traumatic braininjury; Tremor; Trigeminal neuralgia; Tropical spastic paraparesis;Trypanosomiasis; Tuberous sclerosis; Von Hippel-Lindau disease; ViliuiskEncephalomyelitis; Wallenberg's syndrome; Werdnig-Hoffman disease; Westsyndrome; Whiplash; Williams syndrome; Wilson's disease; and Zellwegersyndrome.

In other preferred embodiments of aspects of the invention thecardiovascular disease is selected from the group consisting ofAneurysm; Angina; Atherosclerosis; Cerebrovascular Accident (Stroke);Cerebrovascular disease; Congestive Heart Failure; Coronary ArteryDisease; Myocardial infarction (Heart Attack); and Peripheral vasculardisease.

In other preferred embodiments of aspects of the invention thecardiovascular disease is not systemic lupus erythematosus.

In another aspect, the present invention provides a method of diagnosingdisease in a subject using the method of analysing a blood sampleaccording to the invention. Hence, in another preferred embodiment of amethod of the invention, said method of analysing a blood sampleaccording to the invention is part of a method of diagnosing disease ina subject, and wherein the presence of said disease marker in saidanucleated blood cell-extracted nucleic acid fraction is indicative ofsaid subject suffering from said disease.

In another aspect, the present invention provides a method fordetermining the efficacy of a disease treatment in a subject, comprisingthe steps of;

-   -   analysing a blood sample of a subject for the presence of a        disease marker using the method of analysing a blood sample        according to the invention at a first time point to thereby        provide a first value for the level of said disease marker in        said subject;    -   analysing a blood sample of said subject for the presence of a        disease marker using the method of analysing a blood sample        according to the invention at a second time point that is        earlier or later, preferably later, than said first time point,        to thereby provide a second value for the level of said disease        marker in said subject, wherein said subject has been subjected        to a disease treatment between said first and second time point,        and    -   comparing said first and second value to determine the efficacy        of said disease treatment in said subject.

The skilled artisan will understand that treatment prior to the firsttime point and subsequent measurements at a second, later, time pointwithout any disease treatment having occurred between said time points,is included in aspects of the invention for determining the efficacy ofa disease treatment.

In another aspect, the present invention provides a method fordetermining the stage of disease. In order to determine the stage ofdisease, it is beneficial to correlate disease marker values asdetermined by methods of this invention to disease stages. A singlemeasurement of the disease marker may than be compared to one or morereference values to obtain an indication of the stage of the disease.

In another aspect, the present invention provides a method fordetermining the stage of disease in a subject, comprising the steps of;

-   -   analysing a blood sample of a subject for the presence of a        disease marker using the method of analysing a blood sample of a        subject for the presence of a disease marker according to the        present invention to thereby provide a test value for the level        of said disease marker in said subject,    -   providing a reference value for the level of said disease marker        wherein said reference value is correlated to a particular stage        of disease, and    -   comparing said test and reference value to determine the stage        of disease in said subject.

In yet another aspect, the present invention provides a kit of partsadapted for performing a method of the invention as described hereinabove, the kit comprising a packaging material which comprises at leastone of;

-   -   a container for holding anucleated blood cells, preferably        thrombocytes, separated from a blood sample;    -   an agent for extracting nucleic acids from said anucleated blood        cells;    -   an agent for selectively amplifying from said nucleic acids        extracted from said anucleated blood cells a disease-specific        marker as described herein above, such as a disease-specific        mutation in a gene of a nucleated cell of a subject or a        disease-specific expression profile of nucleic acid from a        nucleated cell of said subject, for instance by reverse        transcriptase polymerase chain reaction amplification, and    -   a printed or electronic instruction for performing a method of        the invention as described herein above, the kit further        comprising:    -   a reference for said disease marker, wherein said reference is        indicative for the presence or absence of said disease marker in        said anucleated blood cells-extracted nucleic acid fraction.

In a preferred embodiment of a kit according to the present inventionsaid reference is a reference value for the level of nucleic acidscomprising said disease-specific mutation in anucleated blood cells in ahealthy control subject or in a control subject suffering from disease,or wherein said reference is a reference expression profile, forinstance for a plurality of mRNAs in anucleated blood cells from ahealthy control subject or from a control subject suffering fromdisease.

In another preferred embodiment of a kit according to the presentinvention said agent or instruction is selected from a particle orfluorescent marker-labeled anti-anucleated blood cell antibody(preferably a fluorescent marker-labeled anti-thrombocyte antibody), aninstruction for bead-based anucleated blood cells isolation (preferablythrombocyte isolation), an instruction for FACS sorting of anucleatedblood cells (preferably of thrombocytes), an instruction for anucleatedblood cell (preferably thrombocyte) recovery by centrifugation, ornegative selection of non-anucleated blood cell components (preferablynon-thrombocyte components).

In yet another aspect, the present invention provides a device fordiagnosing disease, the device comprising a support and at least oneagent for specifically determining a level and/or activity of at leastone nucleic acid mutant in a anucleated blood cells sample of thesubject, said agent being attached to said support, and acomputer-readable medium having computer-executable instructions forperforming a method of the invention as described herein above.

In a preferred embodiment of a device according to the presentinvention, said at least one agent is an oligonucleotide probe orsequencing primer.

In a preferred embodiment of a device according to the presentinvention, the device comprises a lateral flow device, a dipstick or acartridge for performing a nucleic acid hybridization reaction betweenan anucleated blood cells-extracted nucleic acid and at least onenucleic acid mutation-specific amplification primer or oligonucleotideprobe, or between an anucleated blood cells-extracted nucleic acid and aplurality of gene-specific amplification primers or oligonucleotideprobes for providing an disease-specific gene expression profile.

DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B and 1C display RNA profiles as analyzed using an AgilentBioanalyzer Picochip (Agilent Technologies, Inc.), with the length ofthe RNA (in number of nucleotides) on the X-axis, and the amount of RNA(in fluorescence units) on the Y-axis. Here depicted, RNA derived frommicrovesicles in the blood serum fraction (FIG. 1A), RNA derived frommicrovesicles in the blood plasma fraction (FIG. 1B) or RNA derived fromthrombocytes (FIG. C). It is shown that 1) RNA is present inmicrovesicles in serum and plasma and in thrombocytes, 2) microvesiclesisolated from plasma samples contain less RNA than microvesiclesisolated from serum samples, and 3) thrombocytes isolated from plasmasamples contain RNAs of various sizes, including important fractions ofrelatively long RNA chains (>200 nucleotides (nt), and even >1000nucleotides).

FIG. 2 displays the findings of tumour derived genetic material found inthrombocytes from patients with brain tumours. Blood samples frompatients (P1-14) were taken (whole blood tube (serum (S)) andanticoagulant-EDTA blood (plasma (P)). From the plasma tube,thrombocytes (T) were collected by centrifugation protocol. As controls,thrombocytes were collected from healthy individuals (C1-6). Somepatients lack the serum sample, indicated by X in FIG. 2, and some havepooled serum and plasma samples indicated by SP in FIG. 2. Using nestedPCR for RNA detection, the mutant EGFRvIII (V3) could be detected inthrombocytes of 4 glioblastoma patients out of 15 (27%) (P4, P5, P9,P10). This is in line with the published literature where mutantEGFRvIII is found in 20% of high grade gliomas (Liu et al. 2005). Theseexperiments do provide the proof of principle that thrombocytes can beused as a biomarker source for the diagnosis of cancer by theidentification of tumour-derived nucleic acids.

FIGS. 3A-3C. (FIG. 3A) U87 glioma-derived microvesicles were labelledwith PKH67 green fluorescent dye and incubated with isolated platelets.After 15 and 60 min of incubation in the presence and absence ofmicrovesicles the platelets were washed and subjected to FACS analysisof PKH67 fluorescence. In addition, the platelets were stained andanalyzed by confocal microscopy to determine microvesicle uptake. RNAwas isolated from RNase-treated platelets after incubation withmicrovesicles under different conditions. RT-PCR was performed to detectEGFRvIII RNA. MV/MVEGFRvIII: microvesicles isolated fromU87/U87-EGFRvIII cells. (FIG. 3B) RNA was isolated from platelets fromhealthy control subjects or glioma patients and subjected to RT-PCRanalysis. Corresponding glioma tissue biopsies served as control.PC=U87-EGFRvIII RNA; NC=H20; nd=not determined; * indicates positivesignal. (FIG. 3C) RNA as in (B) was subjected to gene expression arrays.Heat map of top-30 glioma biomarkers is shown on the left. Individualexpression levels for the top-10 RNAs depicted on the right. Dashedline=BG (background).

FIG. 4. RNA was isolated from platelets from healthy control subjects(n=8) and prostate cancer patients (n=12) and subjected to PCA3, PSA,and GAPDH RT-PCR analysis. * indicates weak positive signal.

FIG. 5 shows the probe sequences used for the detection of the genesdisplayed in FIG. 3C.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term “cancer” refers to a disease or disorderresulting from the proliferation of oncogenically transformed cells.“Cancer” shall be taken to include any one or more of a wide range ofbenign or malignant tumours, including those that are capable ofinvasive growth and metastasis through a human or animal body or a partthereof, such as, for example, via the lymphatic system and/or the bloodstream. As used herein, the term “tumour” includes both benign andmalignant tumours or solid growths, notwithstanding that the presentinvention is particularly directed to the diagnosis or detection ofmalignant tumours and solid cancers. Cancers further include but are notlimited to carcinomas, lymphomas, or sarcomas, such as, for example,ovarian cancer, colon cancer, breast cancer, pancreatic cancer, lungcancer, prostate cancer, urinary tract cancer, uterine cancer, acutelymphatic leukaemia, Hodgkin's disease, small cell carcinoma of thelung, melanoma, neuroblastoma, glioma (e.g. glioblastoma), and softtissue sarcoma, lymphoma, melanoma, sarcoma, and adenocarcinoma. Inpreferred embodiments of aspects of the present invention, thrombocytecancer is disclaimed.

The term “cancer-derived” as used herein refers to origination from acancer or cancer cell.

The term “cancer-derived nucleic acid” shall be taken to mean anynucleic acid that is indicative of cancer in the subject, specificallyand in most preferred embodiments a mutant DNA or RNA indicating thepresence in the cancer of a mutant gene that is expressed by or ispresent in a cancer cell of the subject, of which mutant gene thenucleic acid sequence is altered relative to the normal gene of ahealthy control subject. The term “cancer-derived nucleic acid” shallalso be taken to include (i) a nucleic acid that is produced by,expressed by, or present in a cancer cell but not in a normal healthy(non-cancerous) cell, or whose production or expression is altered(enhanced or reduced) by or in a cancer cell compared to a normal cell;or (ii) a nucleic acid that is produced by, expressed by, or present ina normal cell but not by or in a cancer cell. Hence, the nucleic acidneed not be a mutant nucleic acid having a mutated sequence but may be anormal nucleic acid having a wild-type (non-cancer) sequence, but whoseprofile or expression level is altered in a cancer cell relative to anormal cell. In one preferred embodiment, the cancer-derived nucleicacid is a mutant nucleic acid (DNA, cDNA, or RNA) specific for thecancer, preferably an RNA transcript. In another very preferredembodiment, the cancer-derived nucleic acid is a nucleic acid expressionprofile indicative of being cancer-derived or cancer-specific, asexplained in detail herein.

As used herein the term “cancer marker” refers to in particular to acancer marker gene or a cancer marker gene expression profile. As usedherein, the term “cancer marker gene” refers to a gene whose sequence orexpression level, alone or in combination with other genes, iscorrelated with cancer or prognosis of cancer. The correlation mayrelate to either an increased or decreased expression of the genereflected in an increased or decreased presence of the RNA expressionproduct of said gene in the nucleic acid fraction obtainable fromthrombocytes. For example, the expression of the gene may be indicativeof cancer, or lack of expression of the gene may be correlated with poorprognosis in a cancer patient. In the case of prostate cancer AMACR,PCA3 and PSA are suitable cancer markers. In the case of colorectalcancer KRAS mutations are suitable cancer markers. In the case of lungcarcinoma EGFR mutations are suitable cancer markers. In the case ofmelanoma BRAF mutations are suitable cancer markers. In the case ofglioma EGFRvIII mutations are suitable cancer markers. Other suitablecancer markers may be derived from Tables 1 and 2 as provided herein orfrom the Examples or Figures. The skilled person will understand thatmany other cancer markers may be employed in aspects and embodiments ofthis invention.

As used herein, the term “stage of cancer” refers to a qualitative orquantitative assessment of the level of advancement of a cancer.Criteria used to determine the stage of a cancer include, but are notlimited to, the size of the tumor, whether the tumor has spread to otherparts of the body and where the cancer has spread (e.g., within the sameorgan or region of the body or to another organ).

The term “cancer” in the terms “cancer derived”, “cancer marker”,“cancer marker gene”, and/or “stage of cancer” may be generalized to theterm “disease” as the definitions for cancer are generally applicable toall diseases as indicated herein.

The term “disease-derived” as used herein refers to origination from adisease or diseased cell.

The term “disease-derived nucleic acid” shall be taken to mean anynucleic acid that is indicative of a disease in the subject,specifically and in most preferred embodiments a mutant DNA or RNAindicating the presence in the disease of a mutant gene that isexpressed by or is present in a diseased cell of the subject, of whichmutant gene the nucleic acid sequence is altered relative to the normalgene of a healthy control subject. The term “disease-derived nucleicacid” shall also be taken to include (i) a nucleic acid that is producedby, expressed by, or present in a diseased cell but not in a normalhealthy (non-diseased) cell, or whose production or expression isaltered (enhanced or reduced) by or in a diseased cell compared to anormal cell; or (ii) a nucleic acid that is produced by, expressed by,or present in a normal cell but not by or in a diseased cell. Hence, thenucleic acid need not be a mutant nucleic acid having a mutated sequencebut may be a normal nucleic acid having a wild-type (non-disease)sequence, but whose profile or expression level is altered in a diseasedcell relative to a normal cell. In one preferred embodiment, thedisease-derived nucleic acid is a mutant nucleic acid (DNA, cDNA, orRNA) specific for the disease, preferably an RNA transcript. In anothervery preferred embodiment, the disease-derived nucleic acid is a nucleicacid expression profile indicative of being disease-derived ordisease-specific, as explained in detail herein. In a preferredembodiment disease-derived nucleic acid does not include cancer-derivednucleic acid. In yet another preferred embodiment, the disease derivednucleic acid does not include vascular disease derived nucleic acid,and/or systemic lupus erythematosus derived nucleic acid. In a preferredembodiment disease-derived nucleic acid does not include sickle celldisease derived nucleic acid. In a preferred embodiment disease-derivednucleic acid does not include Alzheimer's disease derived nucleic acid.In a preferred embodiment of the present invention and embodimentsthereof the disease-derived nucleic acid does not include CD109 nucleicacid. In yet another preferred embodiment of the present invention andembodiments thereof, the disease-derived nucleic acid does not comprisemegakaryocyte derived nucleic acid. In yet another preferred embodimentof the present invention and embodiments thereof, the disease-derivednucleic acid does not comprise nucleic acid derived from diseaseassociated with pathological megakaryocyte and/or platelet function.

As used herein the term “disease marker” refers to in particular to adisease marker gene or a disease marker gene expression profile. As usedherein, the term “disease marker gene” refers to a gene whose sequenceor expression level, alone or in combination with other genes, iscorrelated with disease or prognosis of the disease. The correlation mayrelate to either an increased or decreased expression of the genereflected in an increased or decreased presence of the RNA expressionproduct of said gene in the nucleic acid fraction obtainable fromthrombocytes. For example, the expression of the gene may be indicativeof a disease, or lack of expression of the gene may be correlated withpoor prognosis in a patient. In a preferred embodiment said diseasemarker gene is not a CD109 gene.

As used herein, the term “stage of disease” refers to a qualitative orquantitative assessment of the level of advancement of a disease.Criteria used to determine the stage of a disease include, but are notlimited to, whether the disease has spread to other parts of the bodyand where the disease has spread to (e.g., within the same organ orregion of the body or to another organ).

The term “disease” as used herein may refer to cancer, autoimmunedisease, skin diseases, eye disease, endocrine diseases, neurologicaldisorders, and cardiovascular diseases.

The term “disease” as used herein may refer to autoimmune disease, skindiseases, eye disease, endocrine diseases, neurological disorders,and/or cardiovascular diseases.

The term “disease” as used herein may refer to autoimmune disease, skindiseases, eye disease, endocrine diseases, and/or neurologicaldisorders.

The term “disease” as used herein may, in some preferred embodiments,not refer to cancer, cardiovascular disease, systemic lupuserythematosus, sickle cell disease, Alzheimer's disease, diseasesassociated with pathological platelet function, and/or diseasesassociated with pathological megakaryocyte function.

Thus, diseases that in addition to or instead of cancer can be detectedusing the means and methods of the present invention include forinstance the following auto-immune diseases: Achlorhydra AutoimmuneActive Chronic Hepatitis; Acute Disseminated Encephalomyelitis; Acutehemorrhagic leukoencephalitis; Addison's Disease; Agammaglobulinemia;Alopecia areata; Amyotrophic Lateral Sclerosis; Ankylosing Spondylitis;Anti-GBM/TBM Nephritis; Antiphospholipid syndrome; Antisynthetasesyndrome; polyarticular Arthritis; Atopic allergy; Atopic Dermatitis;Autoimmune Aplastic Anemia; Autoimmune cardiomyopathy; Autoimmuneenteropathy; Autoimmune hemolytic anemia; Autoimmune hepatitis;Autoimmune inner ear disease; Autoimmune lymphoproliferative syndrome;Autoimmune peripheral neuropathy; Autoimmune pancreatitis; Autoimmunepolyendocrine syndrome; Autoimmune progesterone dermatitis; Autoimmunethrombocytopenic purpura; Autoimmune uveitis; Balo disease/Baloconcentric sclerosis; Bechets Syndrome; Berger's disease; Bickerstaffsencephalitis; Blau syndrome; Bullous Pemphigoid; Castleman's disease;Celiac disease; Chagas disease; Chronic Fatigue Immune DysfunctionSyndrome; Chronic inflammatory demyelinating polyneuropathy; Chronicrecurrent multifocal osteomyelitis; Chronic lyme disease; Chronicobstructive pulmonary disease; Churg-Strauss syndrome; CicatricialPemphigoid; Coeliac Disease; Cogan syndrome; Cold agglutinin disease;Complement component 2 deficiency; Cranial arteritis; CREST syndrome;Crohns Disease; Cushing's Syndrome; Cutaneous leukocytoclastic angiitis;Dego's disease; Dercum's disease; Dermatitis herpetiformis;Dermatomyositis; Diabetes mellitus type 1; Diffuse cutaneous systemicsclerosis; Dressler's syndrome; Discoid lupus erythematosus; Eczema;Endometriosis; Enthesitis-related arthritis; Eosinophilic fasciitis;Eosinophilic gastroenteritis; Epidermolysis bullosa acquisita; Erythemanodosum; Essential mixed cryoglobulinemia; Evan's syndrome;Fibrodysplasia ossificans progressiva; Fibromyalgia/Fibromyositis;Fibrosing aveolitis; Gastritis; Gastrointestinal pemphigoid; Giant cellarteritis; Glomerulonep hritis; Goodpasture's syndrome; Graves' disease;Guillain-Barre syndrome; Hashimoto's encephalitis; Hashimoto'sthyroiditis; Haemolytic anaemia; Henoch-Schonlein purpura; Herpesgestationis; Hidradenitis suppurativa; Hughes syndrome;Hypogammaglobulinemia; Idiopathic Inflammatory Demyelinating Diseases;Idiopathic pulmonary fibrosis; Idiopathic thrombocytopenic purpura; IgAnephropathy; Inclusion body myositis; Inflammatory demyelinatingpolyneuopathy; Interstitial cystitis; Irritable Bowel Syndrome (IBS);Juvenile idiopathic arthritis; Juvenile rheumatoid arthritis; Kawasaki'sDisease; Lambert-Eaton myasthenic syndrome; Leukocytoclastic vasculitis;Lichen planus; Lichen sclerosus; Linear IgA disease; Lou Gehrig'sDisease; Lupoid hepatitis; Lupus erythematosus; Majeed syndrome;Meniere's disease; Microscopic polyangiitis; Miller-Fisher syndrome;Mixed Connective Tissue Disease; Morphea; Mucha-Habermann disease;Muckle-Wells syndrome; Multiple Myeloma; Multiple Sclerosis; Myastheniagravis; Myositis; Narcolepsy; Neuromyelitis optica; Neuromyotonia;Occular cicatricial pemphigoid; Opsoclonus myoclonus syndrome; Ordthyroiditis; Palindromic rheumatism; PANDAS; Paraneoplastic cerebellardegeneration; Paroxysmal nocturnal hemoglobinuria; Parry Rombergsyndrome; Parsonnage-Turner syndrome; Pars planitis; Pemphigus;Pemphigus vulgaris; Pernicious anaemia; Perivenous encephalomyelitis;POEMS syndrome; Polyarteritis nodosa; Polymyalgia rheumatica;Polymyositis; Primary biliary cirrhosis; Primary sclerosing cholangitis;Progressive inflammatory neuropathy; Psoriasis; Psoriatic Arthritis;Pyoderma gangrenosum; Pure red cell aplasia; Rasmussen's encephalitis;Raynaud phenomenon; Relapsing polychondritis; Reiter's syndrome;Restless leg syndrome; Retroperitoneal fibrosis; Rheumatoid arthritis;Rheumatoid fever; Sarcoidosis; Schizophrenia; Schmidt syndrome;Schnitzler syndrome; Scleritis; Scleroderma; Sjögren's syndrome;Spondyloarthropathy; Sticky blood syndrome; Still's Disease; Stiffperson syndrome; Subacute bacterial endocarditis (SBE); Susac'ssyndrome; Sweet syndrome; Sydenham Chorea; Sympathetic ophthalmia;Takayasu's arteritis; Temporal arteritis; Tolosa-Hunt syndrome;Transverse Myelitis; Ulcerative Colitis; Undifferentiated connectivetissue disease; Undifferentiated spondyloarthropathy; Vasculitis;Vitiligo; Wegener's granulomatosis; Wilson's syndrome; andWiskott-Aldrich syndrome.

Apart from the above diseases, aspects of the present invention are alsoapplicable to the prognosis and diagnosis of the following skindiseases: Acneiform eruptions; Autoinflammatory syndromes; Chronicblistering; Conditions of the mucous membranes; Conditions of the skinappendages; Conditions of the subcutaneous fat; Congenital anomalies;Connective tissue diseases (such as Abnormalities of dermal fibrous andelastic tissue); Dermal and subcutaneous growths; Dermatitis (includingAtopic Dermatitis, Contact Dermatitis, Eczema, Pustular Dermatitis, andSeborrheic Dermatitis); Disturbances of pigmentation; Drug eruptions;Endocrine-related skin disease; Eosinophilic; Epidermal nevi, neoplasms,cysts; Erythemas; Genodermatoses; Infection-related skin disease;Lichenoid eruptions; Lymphoid-related skin disease; Melanocytic nevi andneoplasms (including Melanoma); Monocyte- and macrophage-related skindisease; Mucinoses; Neurocutaneous; Noninfectiousimmunodeficiency-related skin disease; Nutrition-related skin disease;Papulosquamous hyperkeratotic (including Palmoplantar keratodermas);Pregnancy-related skin disease; Pruritic; Psoriasis; Reactiveneutrophilic; Recalcitrant palmoplantar eruptions; Resulting from errorsin metabolism; Resulting from physical factors (including Ionizingradiation-induced); Urticaria and angioedema; Vascular-related skindisease.

Apart from the above diseases, aspects of the present invention are alsoapplicable to the prognosis and diagnosis of the following endocrinediseases: Adrenal disorders; Glucose homeostasis disorders; Thyroiddisorders; Calcium homeostasis disorders and Metabolic bone disease;Pituitary gland disorders; and Sex hormone disorders.

Apart from the above diseases, aspects of the present invention are alsoapplicable to the prognosis and diagnosis of the following eye diseases:H00-H06 Disorders of eyelid, lacrimal system and orbit; H10-H13Disorders of conjunctiva; H15-H22 Disorders of sclera, cornea, iris andciliary body; H25-H28 Disorders of lens; H30-H36 Disorders of choroidand retina (including H30 Chorioretinal inflammation, H31 Otherdisorders of choroid, H32 Chorioretinal disorders in diseases classifiedelsewhere, H33 Retinal detachments and breaks, H34 Retinal vascularocclusions, H35 Other retinal disorders, and H36 Retinal disorders indiseases classified elsewhere); H40-H42 Glaucoma; H43-H45 Disorders ofvitreous body and globe; H46-H48 Disorders of optic nerve and visualpathways; H49-H52 Disorders of ocular muscles, binocular movement,accommodation and refraction; H53-H54.9 Visual disturbances andblindness; and H55-H59 Other disorders of eye and adnexa.

Apart from the above diseases, aspects of the present invention are alsoapplicable to the prognosis and diagnosis of the following neurologicaldisorders: Abarognosis; Acquired Epileptiform Aphasia; Acutedisseminated encephalomyelitis; Adrenoleukodystrophy; Agenesis of thecorpus callosum; Agnosia; Aicardi syndrome; Alexander disease; Alienhand syndrome; Allochiria; Alpers' disease; Alternating hemiplegia;Alzheimer's disease; Amyotrophic lateral sclerosis (see Motor NeuroneDisease); Anencephaly; Angelman syndrome; Angiomatosis; Anoxia; Aphasia;Apraxia; Arachnoid cysts; Arachnoiditis; Arnold-Chiari malformation;Arteriovenous malformation; Ataxia Telangiectasia; Attention deficithyperactivity disorder; Auditory processing disorder; AutonomicDysfunction; Back Pain; Batten disease; Behcet's disease; Bell's palsy;Benign Essential Blepharospasm; Benign Intracranial Hypertension;Bilateral frontoparietal polymicrogyria; Binswanger's disease;Blepharospasm; Bloch-Sulzberger syndrome; Brachial plexus injury; Brainabscess; Brain damage; Brain injury; Brain tumor; Brown-Séquardsyndrome; Canavan disease; Carpal tunnel syndrome; Causalgia; Centralpain syndrome; Central pontine myelinolysis; Centronuclear myopathy;Cephalic disorder; Cerebral aneurysm; Cerebral arteriosclerosis;Cerebral atrophy; Cerebral gigantism; Cerebral palsy; Cerebralvasculitis; Cervical spinal stenosis; Charcot-Marie-Tooth disease;Chiari malformation; Chorea; Chronic fatigue syndrome; Chronicinflammatory demyelinating polyneuropathy (CIDP); Chronic pain; CoffinLowry syndrome; Coma; Complex regional pain syndrome; Compressionneuropathy; Congenital facial diplegia; Corticobasal degeneration;Cranial arteritis; Craniosynostosis; Creutzfeldt-Jakob disease;Cumulative trauma disorders; Cushing's syndrome; Cytomegalic inclusionbody disease (CIBD); Cytomegalovirus Infection; Dandy-Walker syndrome;Dawson disease; De Morsier's syndrome; Dejerine-Klumpke palsy;Dejerine-Sottas disease; Delayed sleep phase syndrome; Dementia;Dermatomyositis; Developmental dyspraxia; Diabetic neuropathy; Diffusesclerosis; Dravet syndrome; Dysautonomia; Dyscalculia; Dysgraphia;Dyslexia; Dystonia; Empty sella syndrome; Encephalitis; Encephalocele;Encephalotrigeminal angiomatosis; Encopresis; Epilepsy; Erb's palsy;Erythromelalgia; Essential tremor; Fabry's disease; Fahr's syndrome;Fainting; Familial spastic paralysis; Febrile seizures; Fisher syndrome;Friedreich's ataxia; Fibromyalgia; Gaucher's disease; Gerstmann'ssyndrome; Giant cell arteritis; Giant cell inclusion disease; GloboidCell Leukodystrophy; Gray matter heterotopia; Guillain-Barré syndrome;HTLV-1 associated myelopathy; Hallervorden-Spatz disease; Head injury;Headache; Hemifacial Spasm; Hereditary Spastic Paraplegia; Heredopathiaatactica polyneuritiformis; Herpes zoster oticus; Herpes zoster;Hirayama syndrome; Holoprosencephaly; Huntington's disease;Hydranencephaly; Hydrocephalus; Hypercortisolism; Hypoxia;Immune-Mediated encephalomyelitis; Inclusion body myositis;Incontinentia pigmenti; Infantile phytanic acid storage disease;Infantile Refsum disease; Infantile spasms; Inflammatory myopathy;Intracranial cyst; Intracranial hypertension; Joubert syndrome; Karaksyndrome; Kearns-Sayre syndrome; Kennedy disease; Kinsbourne syndrome;Klippel Feil syndrome; Krabbe disease; Kugelberg-Welander disease; Kuru;Lafora disease; Lambert-Eaton myasthenic syndrome; Landau-Kleffnersyndrome; Lateral medullary (Wallenberg) syndrome; Learningdisabilities; Leigh's disease; Lennox-Gastaut syndrome; Lesch-Nyhansyndrome; Leukodystrophy; Lewy body dementia; Lissencephaly; Locked-Insyndrome; Lou Gehrig's disease (See Motor Neurone Disease); Lumbar discdisease; Lumbar spinal stenosis; Lyme disease—Neurological Sequelae;Machado-Joseph disease (Spinocerebellar ataxia type 3); Macrencephaly;Macropsia; Megalencephaly; Melkersson-Rosenthal syndrome; Menieresdisease; Meningitis; Menkes disease; Metachromatic leukodystrophy;Microcephaly; Micropsia; Migraine; Miller Fisher syndrome; Mini-stroke(transient ischemic attack); Mitochondrial myopathy; Mobius syndrome;Monomelic amyotrophy; Motor Neurone Disease; Motor skills disorder;Moyamoya disease; Mucopolysaccharidoses; Multi-infarct dementia;Multifocal motor neuropathy; Multiple sclerosis; Multiple systematrophy; Muscular dystrophy; Myalgic encephalomyelitis; Myastheniagravis; Myelinoclastic diffuse sclerosis; Myoclonic Encephalopathy ofinfants; Myoclonus; Myopathy; Myotubular myopathy; Myotonia congenita;Narcolepsy; Neurofibromatosis; Neurolep tic malignant syndrome;Neurological manifestations of AIDS; Neurological sequelae of lupus;Neuromyotonia; Neuronal ceroid lipofuscinosis; Neuronal migrationdisorders; Niemann-Pick disease; Non 24-hour sleep-wake syndrome;Nonverbal learning disorder; O'Sullivan-McLeod syndrome; OccipitalNeuralgia; Occult Spinal Dysraphism Sequence; Ohtahara syndrome;Olivopontocerebellar atrophy; Opsoclonus myoclonus syndrome; Opticneuritis; Orthostatic Hypotension; Overuse syndrome; Palinopsia;Paresthesia; Parkinson's disease; Paramyotonia Congenita; Paraneoplasticdiseases; Paroxysmal attacks; Parry-Romberg syndrome;Pelizaeus-Merzbacher disease; Periodic Paralyses; Peripheral neuropathy;Persistent Vegetative State; Pervasive developmental disorders; Photicsneeze reflex; Phytanic acid storage disease; Pick's disease; Pinchednerve; Pituitary tumors; PMG; Polio; Polymicrogyria; Polymyositis;Porencephaly; Post-Polio syndrome; Postherpetic Neuralgia (PHN);Postinfectious Encephalomyelitis; Postural Hypotension; Prader-Willisyndrome; Primary Lateral Sclerosis; Prion diseases; Progressivehemifacial atrophy; Progressive multifocal leukoencephalopathy;Progressive Supranuclear Palsy; Pseudotumor cerebri; Rabies; Ramsay-Huntsyndrome (Type I and Type II); Rasmussen's encephalitis; Reflexneurovascular dystrophy; Refsum disease; Repetitive motion disorders;Repetitive stress injury; Restless legs syndrome; Retrovirus-associatedmyelopathy; Rett syndrome; Reye's syndrome; Rhythmic Movement Disorder;Romberg syndrome; Saint Vitus dance; Sandhoff disease; Schizophrenia;Schilder's disease; Schizencephaly; Sensory integration dysfunction;Septo-optic dysplasia; Shaken baby syndrome; Shingles; Shy-Dragersyndrome; Sjögren's syndrome; Sleep apnea; Sleeping sickness;Snatiation; Sotos syndrome; Spasticity; Spina bifida; Spinal cordinjury; Spinal cord tumors; Spinal muscular atrophy; Spinocerebellarataxia; Steele-Richardson-Olszewski syndrome; Stiff-person syndrome;Stroke; Sturge-Weber syndrome; Subacute sclerosing panencephalitis;Subcortical arteriosclerotic encephalopathy; Superficial siderosis;Sydenham's chorea; Syncope; Synesthesia; Syringomyelia; Tarsal tunnelsyndrome; Tardive dyskinesia; Tarlov cyst; Tay-Sachs disease; Temporalarteritis; Tetanus; Tethered spinal cord syndrome; Thomsen disease;Thoracic outlet syndrome; Tic Douloureux; Todd's paralysis; Tourettesyndrome; Toxic encephalopathy; Transient ischemic attack; Transmissiblespongiform encephalopathies; Transverse myelitis; Traumatic braininjury; Tremor; Trigeminal neuralgia; Tropical spastic paraparesis;Trypanosomiasis; Tuberous sclerosis; Von Hippel-Lindau disease; ViliuiskEncephalomyelitis; Wallenberg's syndrome; Werdnig-Hoffman disease; Westsyndrome; Whiplash; Williams syndrome; Wilson's disease; and Zellwegersyndrome.

Apart from the above diseases, aspects of the present invention are alsoapplicable to the prognosis and diagnosis of the followingcardiovascular diseases: Aneurysm; Angina; Atherosclerosis;Cerebrovascular Accident (Stroke); Cerebrovascular disease; CongestiveHeart Failure; Coronary Artery Disease; Myocardial infarction (HeartAttack); and Peripheral vascular disease. In a preferred embodiment ofthe method of the invention and embodiments thereof, and in preferredembodiments of other aspects of the invention, the disease or thecardiovascular disease is not systemic lupus erythematosus.

In a preferred embodiment of the method of the invention and embodimentsthereof, and in preferred embodiments of other aspects of the invention,the disease is not a disease selected from the group comprising cancer,cardiovascular disease, systemic lupus erythematosus, sickle celldisease, Alzheimer's disease, diseases associated with pathologicalplatelet function, and/or diseases associated with pathologicalmegakaryocyte function.

As used herein, “nucleic acid” includes reference to adeoxyribonucleotide or ribonucleotide polymer in either single- ordouble-stranded form, and unless otherwise limited, encompasses knownanalogues having the essential nature of natural nucleotides in thatthey hybridize to single-stranded nucleic acids in a manner similar tonaturally occurring nucleotides (e.g., peptide nucleic acids).

The term “RNA” refers to ribonucleic acid, a molecule of RNA encodingfor a protein product or non-coding for a protein product (such asmiRNAs but not excluding other non-coding RNAs). RNA is transcribed froma DNA template.

As used herein the term “mutant” refers to a nucleic acid compound,protein, molecule, vector or cell resulting from mutation of the nativewild type coding sequence or subunits thereof.

As used herein the term “mutation” refers to any change that alters anative coding sequence either by displacement, addition, deletion,insertion, cross-linking, or other destruction or substitution of one ormore nucleotides of the native coding sequence, including naturallyoccurring splice variants. In particular, the mutation provides a genethat causes the cell to be a cancer cell. Such mutations includeinherited and acquired mutations of tumor suppressor genes and/oroncogenes.

By “amplified” is meant the construction of multiple copies of a nucleicacid sequence or multiple copies complementary to the nucleic acidsequence using at least one of the nucleic acid sequences as a template.Amplification systems include the polymerase chain reaction (PCR)system, ligase chain reaction (LCR) system, nucleic acid sequence basedamplification (NASBA, Cangene, Mississauga, Ontario), Q-Beta Replicasesystems, transcription-based amplification system (TAS), and stranddisplacement amplification (SDA). See, e.g., Diagnostic MolecularMicrobiology. Principles and Applications, D. H. Persing et al., Ed.,American Society for Microbiology, Washington, D.C. (1993). The productof amplification is termed an amplicon.

The term “hybrid” refers to a double-stranded nucleic acid molecule, orduplex, formed by hydrogen bonding between complementary nucleotides.The terms “hybridise” or “anneal” refer to the process by which singlestrands of nucleic acid sequences form double-helical segments throughhydrogen bonding between complementary nucleotides.

The term “oligonucleotide” refers to a short sequence of nucleotidemonomers (usually 6 to 100 nucleotides) joined by phosphorous linkages(e.g., phosphodiester, alkyl and aryl-phosphate, phosphorothioate), ornon-phosphorous linkages (e.g., peptide, sulfamate and others). Anoligonucleotide may contain modified nucleotides having modified bases(e.g., 5-methyl cytosine) and modified sugar groups (e.g., 2′O-methylribosyl, 2′-O-methoxyethyl ribosyl, 2′-fluoro ribosyl, 2′-amino ribosyl,and the like). Oligonucleotides may be naturally-occurring or syntheticmolecules of double- and single-stranded DNA and double- andsingle-stranded RNA with circular, branched or linear shapes andoptionally including domains capable of forming stable secondarystructures (e.g., stem-and-loop and loop-stem-loop structures).

The term “primer” as used herein refers to an oligonucleotide which iscapable of annealing to the amplification target allowing a DNApolymerase to attach thereby serving as a point of initiation of DNAsynthesis when placed under conditions in which synthesis of primerextension product which is complementary to a nucleic acid strand isinduced, i.e., in the presence of nucleotides and an agent forpolymerization such as DNA polymerase and at a suitable temperature andpH. The (amplification) primer is preferably single stranded for maximumefficiency in amplification. Preferably, the primer is an oligodeoxyribonucleotide. The primer must be sufficiently long to prime thesynthesis of extension products in the presence of the agent forpolymerization. The exact lengths of the primers will depend on manyfactors, including temperature and source of primer. A “pair ofbi-directional primers” as used herein refers to one forward and onereverse primer as commonly used in the art of DNA amplification such asin PCR amplification.

The term “probe” refers to a single-stranded oligonucleotide sequencethat will recognize and form a hydrogen-bonded duplex with acomplementary sequence in a target nucleic acid sequence analyte or itscDNA derivative.

The terms “stringency” or “stringent hybridization conditions” refer tohybridization conditions that affect the stability of hybrids, e.g.,temperature, salt concentration, pH, formamide concentration and thelike. These conditions are empirically optimised to maximize specificbinding and minimize non-specific binding of primer or probe to itstarget nucleic acid sequence. The terms as used include reference toconditions under which a probe or primer will hybridise to its targetsequence, to a detectably greater degree than other sequences (e.g. atleast 2-fold over background). Stringent conditions are sequencedependent and will be different in different circumstances. Longersequences hybridise specifically at higher temperatures. Generally,stringent conditions are selected to be about 5° C. lower than thethermal melting point (T_(m)) for the specific sequence at a definedionic strength and pH. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of a complementary target sequencehybridises to a perfectly matched probe or primer. Typically, stringentconditions will be those in which the salt concentration is less thanabout 1.0 M Na⁺ ion, typically about 0.01 to 1.0 M Na⁺ ion concentration(or other salts) at pH 7.0 to 8.3 and the temperature is at least about30° C. for short probes or primers (e.g. 10 to 50 nucleotides) and atleast about 60° C. for long probes or primers (e.g. greater than 50nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringent conditions or “conditions of reduced stringency” includehybridization with a buffer solution of 30% formamide, 1 M NaCl, 1% SDSat 37° C. and a wash in 2×SSC at 40° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a wash in 0.1×SSC at 60° C. Hybridization procedures arewell known in the art and are described in e.g. Ausubel et al, CurrentProtocols in Molecular Biology, John Wiley & Sons Inc., 1994.

“Subject” as used herein includes, but is not limited to, mammals,including, e.g., a human, a non-human primate, a mouse, a pig, a cow, agoat, a cat, a rabbit, a rat, a guinea pig, a hamster, a degu, a horse,a monkey, a sheep, or other non-human mammal; and non-mammal animals,including, e.g., a non-mammalian vertebrate, such as a bird (e.g., achicken or duck) or a fish, and an invertebrate. The subject may be ahealthy animal or human subject undergoing a routine well-being checkup. Alternatively, the subject may be at risk of having a disease (e.g.,a genetically predisposed subject, a subject with medical and/or familyhistory of cancer, a subject who has been exposed to carcinogens,occupational hazard, environmental hazard] and/or a subject who exhibitssuspicious clinical signs of a disease [e.g., blood in the stool ormelena, unexplained pain, sweating, unexplained fever, unexplained lossof weight up to anorexia, changes in bowel habits (constipation and/ordiarrhoea), tenesmus (sense of incomplete defecation, for rectal cancerspecifically), anaemia and/or general weakness). According to anotherembodiment, the subject may be a patient diagnosed with the disease andis performing a routine check-up, in-between treatments.

The term “thrombocyte”, as used herein, refers to blood platelets, i.e.the small, irregularly-shaped cell fragments that do not have a nucleuscontaining DNA, and that circulate in the blood of mammals. Thrombocytesare 2-3 μm in diameter, and are derived from fragmentation of precursormegakaryocytes. Platelets or thrombocytes lack nuclear DNA, althoughthey retain some megakaryocyte-derived mRNAs as part of their linealorigin. The average lifespan of a thrombocyte is 5 to 9 days.Thrombocytes are involved and play an essential role in haemostasis,leading to the formation of blood clots. In a preferred embodiment ofthe present invention and embodiments thereof, the anucleated bloodcell-extracted nucleic acid fraction is not megakaryocyte-derivednucleic acid or megakaryocyte-derived RNA.

The term “blood” as used herein refers to whole blood (including plasmaand cells) and includes arterial, capillary and venous blood.

The term “nucleated cell” as used herein preferably refers to aBartholin's gland cell; Salivary gland mucous cell; Salivary glandserous cell; Von Ebner's gland cell; Mammary gland cell; Lacrimal glandcell; Ceruminous gland cell; Eccrine sweat gland cell; Apocrine sweatgland cell; Gland of Moll cell; Sebaceous gland cell; Bowman's glandcell; Brunner's gland cell; Seminal vesicle cell; Prostate gland cell;Bulbourethral gland cell; Gland of Littre cell; Uterus endometrium cell;Isolated goblet cell; Stomach lining mucous cell; Gastric glandzymogenic cell; Gastric gland oxyntic cell; Pancreatic acinar cell;Paneth cell; Type II pneumocyte; Clara cell; Anterior pituitary cell;Intermediate pituitary cell; Magnocellular neurosecretory cell; Thyroidgland cell; Parathyroid gland cells; Adrenal gland cells; Leydig cell;Theca interna cell; Corpus luteum cell; Juxtaglomerular cell; Maculadensa cell; Peripolar cell; Mesangial cell; Blood vessel and lymphaticvascular endothelial fenestrated cell; Blood vessel and lymphaticvascular endothelial continuous cell; Blood vessel and lymphaticvascular endothelial splenic cell; Synovial cell; Serosal cell; Squamouscell; Columnar cell; Dark cell; Vestibular membrane cell; Striavascularis basal cell; Stria vascularis marginal cell; Cell of Claudius;Cell of Boettcher; Choroid plexus cell; Pia-arachnoid squamous cell;Pigmented ciliary epithelium cell; Nonpigmented ciliary epithelium cell;Corneal endothelial cell; Peg cell; Respiratory tract ciliated cell;Oviduct ciliated cell; Uterine endometrial ciliated cell; Rete testisciliated cell; Ductulus efferens ciliated cell; Ciliated ependymal cell;Epidermal keratinocyte; Epidermal basal cell; Keratinocyte; Nail bedbasal cell; Medullary hair shaft cell; Cortical hair shaft cell;Cuticular hair shaft cell; Cuticular hair root sheath cell; Externalhair root sheath cell; Hair matrix cell; Surface epithelial cell; basalcell; Urinary epithelium cell; Auditory inner hair cell; Auditory outerhair cell; Primary sensory neurons; Merkel cell; Olfactory receptorneuron; Photoreceptor cells; Carotid body cell (blood pH sensor); Haircell; Taste bud cell; Cholinergic neural cell; Adrenergic neural cell;Peptidergic neural cell; Inner pillar cell; Outer pillar cell; Innerphalangeal cell; Outer phalangeal cell; Border cell; Hensen cell;Vestibular apparatus supporting cell; Taste bud supporting cell;Olfactory epithelium supporting cell; Schwann cell; Satellite cell;Enteric glial cell; Astrocyte; Neuron cells; Oligodendrocyte; Spindleneuron; Anterior lens epithelial cell; Crystallin-containing lens fibercell; Hepatocyte; Adipocytes; Liver lipocyte; Kidney glomerulus parietalcell; Kidney glomerulus podocyte; Kidney proximal tubule brush bordercell; Loop of Henle thin segment cell; Kidney distal tubule cell; Kidneycollecting duct cell; pneumocyte; Pancreatic duct cell; Nonstriated ductcell; Duct cell; Intestinal brush border cell; Exocrine gland striatedduct cell; Gall bladder epithelial cell; Ductulus efferens nonciliatedcell; Epididymal principal cell; Epididymal basal cell; Ameloblastepithelial cell; Planum semilunatum epithelial cell; Organ of Cortiinterdental epithelial cell; Loose connective tissue fibroblasts;Corneal fibroblasts; Tendon fibroblasts; Bone marrow reticular tissuefibroblasts; Other nonepithelial fibroblasts; Pericyte; Nucleus pulposuscell; Cementoblast/cementocyte; Odontoblast/odontocyte; Hyalinecartilage chondrocyte; Fibrocartilage chondrocyte; Elastic cartilagechondrocyte; Osteoblast/osteocyte; Osteoprogenitor cell; Hyalocyte;Stellate cell; Hepatic stellate cell; Pancreatic stelle cell; Skeletalmuscle cell; Satellite cell; Heart muscle cell; Smooth muscle cell;Myoepithelial cell; Monocyte; Connective tissue macrophage; EpidermalLangerhans cell; Osteoclast; Dendritic cell; Microglial cell; Neutrophilgranulocyte; Eosinophil granulocyte; Basophil granulocyte; Mast cell;Helper T cell; Suppressor T cell; Cytotoxic T cell; Natural Killer Tcell; B cell; Natural killer cell; Reticulocyte; Melanocyte; Retinalpigmented epithelial cell; Oogonium/Oocyte; Spermatid; Spermatocyte;Spermatogonium cell; Spermatozoon; Ovarian follicle cell; Sertoli cell;Thymus epithelial cell; and Interstitial kidney cell.

Targeted therapy and personalized medicine are critically depending ondisease profiling and the development of companion diagnostics.Mutations in disease-derived nucleic acids can be highly predictive forthe response to targeted treatment. However, obtaining easily accessiblehigh-quality nucleic acids remains a significant developmental hurdle.Blood generally contains 150,000-350,000 thrombocytes (platelets) permicroliter, providing a highly available biomarker source for researchand clinical use. Moreover, thrombocyte isolation is relatively simpleand is a standard procedure in blood bank/haematology labs. Sinceplatelets do not contain a nucleus, their RNA transcripts—needed forfunctional maintenance—are derived from bone marrow megakaryocytesduring thrombocyte origination. It has now been found that thrombocytesmay take up RNA and/or DNA from cells other than megakaryocytes duringcirculation via various transfer mechanisms. Tumor cells for instancerelease an abundant collection of genetic material, some of which issecreted by microvesicles in the form of mutant RNA. During circulationin the blood stream thrombocytes absorb the genetic material secreted bycancer cells and other diseased cells, serving as an attractive platformfor the companion diagnostics of cancer and other diseases as indicatedabove e.g. in the context of personalized medicine.

In the Examples below it is shown that platelets isolated from healthyhuman control subjects have the ability to take up RNA fromRNA-containing microvesicles derived from human brain tumor cells(glioma), after which they contain tumor-associated RNA, including forinstance mutant EGFRvIII mRNA in the case of glioma patients. Hence, itwas determined that circulating platelets isolated from glioma patientscontain RNA biomarkers. RT-PCR was used to confirm that mutant EGFRvIIImRNA found in the thrombocytes reflects the presence of glioma tissues.

The presence of tumor and/or disease-markers messages is not unique toplatelets from glioma patients but is more generally applicable for awide range of diseases as identified herein. Messenger RNAs coding forthe prostate cancer markers PCA3 and PSA could be demonstrated inplatelets from prostate cancer patients, whereas these markers wereabsent in platelets from healthy control subjects.

Apart from detecting gene mutations associated with cancer or otherdiseases, the present inventors also found that gene expression arrayscould be used to classify a thrombocytes nucleic acid sample as beingthat of a subject suffering from a specific type of (solid tumour)cancer or other disease. It was established that mRNA expressionprofiles obtained with nucleic acids extracted from platelets isolatedfrom healthy control subjects or extracted from platelets isolated fromglioma patients differed specifically. Distinct mRNA expression profileswere obtained and a minimal glioma biomarker signature could bedetected, as shown for the Top-30 hits in FIG. 3C. The distinct profileas shown in FIG. 3C comprises a significant increase in the expressionof the following genes: WFDC1, Kremen1, DEF4A, ARG1, FKBP5, ACRC,ENST0328043, A_32_P167111, MAP2, ECTL8, UNC13B, TP53I3, FDXR, BX119718,SORT1, PFN4, C1QTNF5, A_24_P237896, PGLYRP1, SEC14L2, BC018626, MAOB,TCN1, AMOTL1, TSP50, A_24_P927015, THC2325987, C18orf1, and LIN28 (someof these gene names are referred to with reference to the MicroarrayAccession number, e.g. the oligonucleotide probe of the Agilent Chip).It will be understood that this profile is not limitative to the scopeof the present invention, since the skilled person is well aware how toobtain other suitable gene expression profiles using the methods of thepresent invention for other cancers, and for other diseases in general.

The present inventors have now found that blood platelets contain cancermarkers and disease markers in the form of tumor-derived ortumor-associated or disease-derived nucleic acids or nucleic acidexpression profiles and that these platelets may serve as a diagnosticplatform for the molecular profiling of cancer and other diseases asidentified herein. This is highly useful in the context of personalizedmedicine.

The present invention provides a novel and easy-to-use method to isolatecirculating disease-derived material (e.g. disease markers as usedherein) for genetic analysis. The present inventors isolatedtumor-derived RNA from circulating thrombocytes, yielding pure RNA andthereby providing an easy way to extract high quality RNA from lowamounts of blood. Thrombocyte nucleic acid (NA) isolation and subsequentanalysis presents a marked increase in the diagnostic sensitivity ofcirculating NA in blood.

The present inventors found that in diseased patients circulatingthrombocytes contain significant amounts of disease-derived RNA and/orDNA. This disease-derived RNAs and/or DNAs presents unique geneticinformation about the disease, which may be used to determine diseasetype, extent of disease and possibly the susceptibility of the diseaseto therapeutic treatment. In a preferred embodiment of a method orembodiment of the invention said disease is not cancer.

In another preferred embodiment of a method or embodiment of theinvention said disease is not a vascular disease.

In another preferred embodiment of a method or embodiment of theinvention said disease is not systemic lupus erythematosus.

In another preferred embodiment of a method or embodiment of theinvention said disease is not sickle cell disease.

In another preferred embodiment of a method or embodiment of theinvention said disease is not Alzheimer's disease.

In another preferred embodiment of a method or embodiment of theinvention said disease is not a disease associated with pathologicalmegakaryocyte function.

In another preferred embodiment of a method or embodiment of theinvention said disease is not a disease associated with pathologicalplatelet function.

The above-referenced preferred embodiments wherein certain diseases aredisclaimed can be combined in any manner in aspects of this invention.

In a preferred embodiment of the method of the invention and embodimentsthereof the disease is not a disease selected from the group comprisingcancer, cardiovascular disease, systemic lupus erythematosus, sicklecell disease, Alzheimer's disease, diseases associated with pathologicalplatelet function, and/or diseases associated with pathologicalmegakaryocyte function.

Diseases involving abnormal platelet function may comprisepost-transfusion purpura (PTP), post transfusion plateletrefractioriness (PTPR), nenotal alloimmune thrombocytopenia (NATP),thrombocytopenia, and/orthrombocythaemia.

In another preferred embodiment of a method or embodiment of theinvention said disease-derived nucleic acid does not originate from amegakaryocyte. It is expressly indicated that the nucleic acid subjectof this invention is taken up or accumulated by the thrombocyte from theextracellular (blood plasma) environment, and not megakaryocyte-lineagederived. The thrombocyte RNA and/or DNA can be analyzed for the presenceof specific disease-derived RNAs and/or DNAs, as demonstrated herein forthe EGFRvIII mutant RNA derived from glioma tumours.

The present invention describes a method of finding specific nucleicacid transcripts derived from nucleated cells of disease origin withinanucleated blood cells such as thrombocytes extracted from blood. Thisapproach is robust and easy. This is attributed to the rapid andstraight forward extraction procedures and the quality of the extractedNA. Within the clinical setting, thrombocytes extraction (from bloodsamples) is already implemented in general biological sample collectionand therefore it is foreseen that the implementation into the clinic isrelatively easy.

The present invention provides a general method for analysing blood of asubject for the presence of a disease-derived nucleic acid and a methodof diagnosing disease in a subject using said general method. Whenreference is herein made to a method of the invention, both embodimentsare referred to.

A method of the invention can be performed on any suitable body samplecomprising anucleated blood cells, such as for instance a tissue samplecomprising blood, but preferably said sample is whole blood.

A blood sample of a subject can be obtained by any standard method, forinstance by venous extraction.

The amount of blood needed is not particularly limited. Depending on themethods employed, the skilled person will be capable of establishing theamount of sample required to perform the various steps of the method ofthe present invention and obtain sufficient NA for genetic analysis.Generally, such amounts will comprise a volume ranging from 0.01 μl to100 ml.

The body sample may be analyzed immediately following collection of thesample. Alternatively, analysis according to the method of the presentinvention can be performed on a stored body sample or on a storedfraction of anucleated blood cells thereof, preferably thrombocytes. Thebody sample for testing, or the fraction of anucleated blood cellsthereof, can be preserved using methods and apparatuses known in theart. In a collected anucleated blood cell fraction, the thrombocytes arepreferably maintenance in inactivated state (i.e. in non-activatedstate). In that way, the cellular integrity and the disease-derivednucleic acids are best preserved.

In case the fraction of anucleated blood cells is a thrombocytefraction, this platelet isolated fraction does preferably not includeplatelet poor plasma or platelet rich plasma (PRP). Further isolation ofthe platelets is preferred for optimal resolution.

The body sample may suitably be processed otherwise, for instance, itmay be purified, or digested, or specific compounds may be extractedtherefrom. Depending upon the method of characterizing the NA present inthe anucleated blood cells in said sample, which method preferablyinvolves RT-PCR, the anucleated blood cells may be extracted from thesample by methods known to the skilled person and be transferred to anysuitable medium for extraction of the NA therefrom should the analysismethod so require. The recipient subject's body sample may be treated toremove abundant nucleic acid degrading enzymes (like RNases, DNases)therefrom, in order to prevent early destruction of the nucleic acids.

Thrombocyte extraction from the body sample of the subject may involveany available method. In transfusion medicine, thrombocytes are oftencollected by apheresis, a medical technology in which the blood of adonor or patient is passed through an apparatus that separates out oneparticular constituent and returns the remainder to the circulation. Theseparation of individual blood components is done with a specializedcentrifuge. Plateletpheresis (also called thrombopheresis orthrombocytapheresis) is the apheresis process of collectingthrombocytes. Modern automatic plateletpheresis allows blood donors togive a portion of their thrombocytes, while keeping their red bloodcells and at least a portion of blood plasma. Although it is possible toprovide the body sample comprising thrombocytes as envisioned herein byapheresis, it is often easier to collect whole blood and isolate thethrombocyte fraction therefrom by centrifugation. Generally, in such aprotocol, the thrombocytes are first separated from the other bloodcells by a centrifugation step of about 120×g for about 20 minutes atroom temperature to obtain a platelet rich plasma (PRP) fraction. Thethrombocytes are then washed (for instance in PBS-EDTA) to remove plasmaproteins and enrich for thrombocytes. Wash steps are generally carriedout at 850-1000×g for about 10 min at room temperature. Furtherenrichments can be carried out to yield more pure thrombocyte fractions.

Platelet isolation generally involves blood sample collection inVacutainer tubes containing anticoagulant citrate dextrose (e.g. 36 mlcitric acid, 5 mmol/l KCl, 90 mmol/1 NaCl, 5 mmol/1 glucose, 10 mmol/1EDTA pH 6.8). A suitable protocol for platelet isolation is described inFerretti et al. (J Clin Endocrinol Metab 2002; 87:2180-2184). Thismethod involves a preliminary centrifugation step (1,300 rpm per 10 min)to obtain platelet-rich plasma (PRP). Platelets are then washed threetimes in an anti-aggregation buffer (Tris-HCl 10 mmol/1; NaCl 150mmol/1; EDTA 1 mmol/1; glucose 5 mmol/1; pH 7.4) and centrifuged asabove, to avoid any contamination with plasma proteins and to remove anyresidual erythrocytes. A final centrifugation at 4,000 rpm for 20 minmay then be performed to isolate platelets. The platelet pellet may bewashed (e.g. in phosphate buffered saline For quantitative determinationof disease marker levels, the protein concentration of plateletmembranes may be used as internal reference. Such protein concentrationsmay be determined by the method of Bradford (Anal Biochem 1976;72:248-254), using serum albumin as standard.

Following the provision of the body sample of the subject, and theextraction therefrom of the anucleated blood cells, the anucleated bloodcells of the subject are screened for the presence of disease-specificnucleic acids. If disease-specific nucleic acids are encountered in theanucleated blood cells of the subject, or if disease-specific nucleicacids are encountered in the anucleated blood cells of the subject at ahigher level than in the anucleated blood cells in an unaffected bloodsample of a control subject, which disease-specific nucleic acids areconsidered to originate from a diseased cell or tissue residing in thesubject, said subject is diagnosed with disease as defined herein.

Disease-specific nucleic acids (RNA and/or DNA disease markers) aredefined as originating from disease cells that contain mutations or nomutations in the nucleic acid sequences that are associated with orspecific to the disease, and also include disease-derived anucleatedblood cells nucleic acids which are up- or down-regulated as compared tonucleic acids in anucleated blood cells from healthy donors. Hence, theterms “disease-specific nucleic acids” and “disease-derived nucleicacids” are used interchangeable herein. It will be appreciated thatnon-mutated genes can be identified and used for disease diagnostics. Ifcertain genes are overexpressed in certain diseases, these nucleic acidsmay be transferred to anucleated blood cells. However, if these nucleicacids are already present in anucleated blood cells of healthy subjectsone can expect an increase in the number of nucleic acid copies inanucleated blood cells of such diseased patients. Hence, quantificationof the copy number of certain genes (by quantitative PCR or microarrayse.g.) in anucleated blood cells may be beneficial in certain embodimentsof aspects of this invention for detecting the presence of a diseasesoverexpressing such genes. Preferably the disease marker ordisease-specific nucleic acids are not derived from a megakaryocyte. Ina preferred embodiment of the present invention and embodiments thereofthe disease marker or disease specific nucleic acid is not a mutation atposition 12027 in mitochondrial DNA. In a preferred embodiment of thepresent invention and embodiments thereof the disease marker or diseasespecific nucleic acid is not a mutation at position 11778 inmitochondrial DNA. In a preferred embodiment of the present inventionand embodiments thereof the disease marker or disease specific nucleicacid is not a mutation in the CD109 gene. In a preferred embodiment ofthe present invention and embodiments thereof the disease marker ordisease specific nucleic acid is not a mutation at position 2108 and/orposition 954 of the coding region of the CD109 gene. Any of the abovedisclaimed embodiments may be disclaimed in any combination in aspectsherein.

A further step in a method of the invention is the provision of ananucleated blood cells-extracted nucleic acid fraction. Such a nucleicacid fraction is subsequently used for the detection of a disease markertherein. An anucleated blood cells-extracted nucleic acid fraction maybe obtained by any NA extraction method available. Usually RNAextraction is performed by using chaotropic reagents. The first step inisolating total RNA from cells or tissue is to break open the cellsunder denaturing conditions. In 1979, Chirgwin et al. (Biochemistry,18[24]:5294-9, 1979) devised a method for the efficient isolation oftotal RNA by homogenization in a 4 M solution of the potent proteindenaturant guanidinium thiocyanate with 0.1 M 2-mercaptoethanol to breakprotein disulfide bonds. RNA was then isolated by ethanol extraction orby ultracentrifugation through cesium chloride. In 1987 Chomczynski andSacchi (Analytical Biochemistry, 162[1]:156-9, 1987) modified thismethod to devise a rapid single-step extraction procedure using amixture of guanidinium thiocyanate and phenol-chloroform, a methodespecially useful for processing large numbers of samples or forisolation of RNA from small quantities of cells or tissue. Anycommercial kit can also be used for the extraction of RNA, non-limitingexamples thereof include Ambion's RNAqueous™ system, Bio101's RNaid Pluskit, Bioline Ltd.'s RNAce kits, CLONTECH's NucleoSpin® RNA II andNucleoTrap mRNA kits, Invitrogen Corp.'s S.N.A.P. Total RNA IsolationKit and QIAGEN's RNeasy kits.

The detection of a disease-derived nucleic acid in the extracted nucleicacid sample may occur by any genetic analysis technique available thatis suitable for the detection of nucleic acid sequence mutations orexpression profiles in nucleic acids that are specific for the disease.Usually, such sequence mutations can be easily detected by selectivenucleic acid hybridization, involving the formation of a duplex nucleicacid structure formed by selective hybridization with each other of twosingle-stranded nucleic acid sequences. Selective hybridization includesreference to hybridization, under stringent hybridization conditions, ofa nucleic acid sequence to a specified nucleic acid target sequence to adetectably greater degree (e.g., at least 2-fold over background) thanits hybridization to non-target nucleic acid sequences and to thesubstantial exclusion of non-target nucleic acids. Selectivelyhybridizing sequences typically have about at least 80% sequenceidentity, preferably 90% sequence identity, and most preferably 100%sequence identity (i.e., complementary) with each other.

Alternatively, detection of a disease-derived nucleic acid may occurthrough sequencing technologies such as DNA and RNA sequencing.

When detecting sequence mutations in RNA, or expression profiles of RNA,it is preferred that the RNA is transcribed into cDNA prior to thedetection of sequence mutations therein or quantitation of the amountexpressed.

RNA can be reverse transcribed into cDNA using RNA-dependent DNApolymerases such as, for example, reverse transcriptases from viruses,retrotransposons, bacteria, etc. These can have RNase H activity, orreverse transcriptases can be used that are so mutated that the RNase Hactivity of the reverse transcriptase was restricted or is not present(e.g. MMLV-RT RNase H⁻). RNA-dependent DNA synthesis (reversetranscription) can also be carried by enzymes that show altered nucleicacid dependency through mutation or modified reaction conditions andthus obtain the function of the RNA-dependent DNA polymerase. Commercialkits are available to reverse transcribe RNA into cDNA.

Once the RNA is reverse transcribed into cDNA, the DNA sequence can beanalysed for the presence of cancer-specific mutations or expressionprofiles can be determined using for instance selective nucleic acidhybridization as described above. Such techniques are well known in theart and may comprise selective amplification using amplification primersthat are specific for the mutation to be detected or selectivehybridization to nucleic acid arrays using mRNA-specific probes.Alternatively, general primers can be used to amplify the DNA comprisingthe suspected mutation and the mutation can than be detected in theamplicon by selective nucleic acid hybridization using probes that arespecific for the mutation. Expression profiles are generally obtainedusing methods of quantitative hybridization well described in the art,an illustration of which is described in the Examples. Methods of theinvention can in principle be performed by using any nucleic acidamplification method, such as the Polymerase Chain Reaction (PCR; Mullis1987, U.S. Pat. Nos. 4,683,195, 4,683,202, en 4,800,159) or by usingamplification reactions such as Ligase Chain Reaction (LCR; Barany 1991,Proc. Natl. Acad. Sci. USA 88:189-193; EP Appl. No., 320,308),Self-Sustained Sequence Replication (35R; Guatelli et al., 1990, Proc.Natl. Acad. Sci. USA 87:1874-1878), Strand Displacement Amplification(SDA; U.S. Pat. Nos. 5,270,184, and 5,455,166), TranscriptionalAmplification System (TAS; Kwoh et al., Proc. Natl. Acad. Sci. USA86:1173-1177), Q-Beta Replicase (Lizardi et al., 1988, Bio/Technology6:1197), Rolling Circle Amplification (RCA; U.S. Pat. No. 5,871,921),Nucleic Acid Sequence Based Amplification (NASBA), Cleavase FragmentLength Polymorphism (U.S. Pat. No. 5,719,028), Isothermal and ChimericPrimer-initiated Amplification of Nucleic Acid (ICAN),Ramification-extension Amplification Method (RAM; U.S. Pat. Nos.5,719,028 and 5,942,391) or other suitable methods for amplification ofDNA.

In order to amplify DNA with a small number of mismatches to one or moreof the amplification primers, an amplification reaction may be performedunder conditions of reduced stringency (e.g. a PCR amplification usingan annealing temperature of 38° C., or the presence of 3.5 mM MgCl₂).The person skilled in the art will be able to select conditions ofsuitable stringency.

The primers herein are selected to be “substantially” complementary(i.e. at least 65%, more preferably at least 80% perfectlycomplementary) to their target regions present on the different strandsof each specific sequence to be amplified. It is possible to use primersequences containing e.g. inositol residues or ambiguous bases or evenprimers that contain one or more mismatches when compared to the targetsequence. In general, sequences that exhibit at least 65%, morepreferably at least 80% homology with the target DNA oligonucleotidesequences, are considered suitable for use in a method of the presentinvention. Sequence mismatches are also not critical when using lowstringency hybridization conditions.

The detection of the amplification products can in principle beaccomplished by any suitable method known in the art. The detectionfragments may be directly stained or labelled with radioactive labels,antibodies, luminescent dyes, fluorescent dyes, or enzyme reagents.Direct DNA stains include for example intercalating dyes such asacridine orange, ethidium bromide, ethidium monoazide or Hoechst dyes.

Alternatively, the DNA fragments may be detected by incorporation oflabelled dNTP bases into the synthesized DNA fragments. Detection labelswhich may be associated with nucleotide bases include e.g. fluorescein,cyanine dye or BrdUrd.

When using a probe-based detection system, a suitable detectionprocedure for use in the present invention may for example comprise anenzyme immunoassay (EIA) format (Jacobs et al., 1997, J. Clin.Microbiol. 35, 791795). For performing a detection by manner of the EIAprocedure, either the forward or the reverse primer used in theamplification reaction may comprise a capturing group, such as a biotingroup for immobilization of target DNA PCR amplicons on e.g. astreptavidin coated microtiter plate wells for subsequent EIA detectionof target DNA amplicons (see below). The skilled person will understandthat other groups for immobilization of target DNA PCR amplicons in anEIA format may be employed.

Probes useful for the detection of the target DNA as disclosed hereinpreferably bind only to at least a part of the DNA sequence region asamplified by the DNA amplification procedure. Those of skill in the artcan prepare suitable probes for detection based on the nucleotidesequence of the target DNA without undue experimentation as set outherein. Also the complementary sequences of the target DNA may suitablybe used as detection probes in a method of the invention, provided thatsuch a complementary strand is amplified in the amplification reactionemployed.

Suitable detection procedures for use herein may for example compriseimmobilization of the amplicons and probing the DNA sequences thereof bye.g. southern blotting. Other formats may comprise an EIA format asdescribed above. To facilitate the detection of binding, the specificamplicon detection probes may comprise a label moiety such as afluorophore, a chromophore, an enzyme or a radio-label, so as tofacilitate monitoring of binding of the probes to the reaction productof the amplification reaction. Such labels are well-known to thoseskilled in the art and include, for example, fluorescein isothiocyanate(FITC), β-galactosidase, horseradish peroxidase, streptavidin, biotin,digoxigenin, ³⁵S or ¹²⁵I. Other examples will be apparent to thoseskilled in the art.

Detection may also be performed by a so called reverse line blot (RLB)assay, such as for instance described by Van den Brule et al. (2002, J.Clin. Microbiol. 40, 779-787). For this purpose RLB probes arepreferably synthesized with a 5′amino group for subsequentimmobilization on e.g. carboxylcoated nylon membranes. The advantage ofan RLB format is the ease of the system and its speed, thus allowing forhigh throughput sample processing.

The use of nucleic acid probes for the detection of DNA fragments iswell known in the art. Mostly these procedures comprise thehybridization of the target DNA with the probe followed bypost-hybridization washings. Specificity is typically the function ofpost-hybridization washes, the critical factors being the ionic strengthand temperature of the final wash solution. For DNA-DNA hybrids, theT_(m) (the thermal melting point, i.e. the temperature under definedionic strength and pH at which 50% of a complementary target sequencehybridizes to a perfectly matched probe) can be approximated from theequation of Meinkoth and Wahl (Anal. Biochem., 138: 267-284 (1984)):T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L; where M isthe molarity of monovalent cations, % GC is the percentage of guanosineand cytosine nucleotides in the DNA, % form is the percentage offormamide in the hybridization solution, and L is the length of thehybrid in base pairs. The T_(m) is reduced by about 1° C. for each 1% ofmismatching; thus, the hybridization and/or wash conditions can beadjusted to hybridize to sequences of the desired identity. For example,if sequences with >90% identity are sought, the T_(m) can be decreased10° C. Generally, stringent conditions are selected to be about 5° C.lower than the T_(m) for the specific sequence and its complement at adefined ionic strength and pH. However, severely stringent conditionscan utilize a hybridization and/or wash at 1, 2, 3, or 4° C. lower thanT_(m) moderately stringent conditions can utilize a hybridization and/orwash at 6, 7, 8, 9, or 10° C. lower than the T_(m); low stringencyconditions can utilize a hybridization and/or wash at 11, 12, 13, 14,15, or 20° C. lower than T_(m). Using the equation, hybridization andwash compositions, and desired T_(m), those of ordinary skill willunderstand that variations in the stringency of hybridization and/orwash solutions are inherently described. If the desired degree ofmismatching results in a T_(m) of less than 45° C. (aqueous solution) or32° C. (formamide solution) it is preferred to increase the SSCconcentration so that a higher temperature can be used. An extensiveguide to the hybridization of nucleic acids is found in Tijssen,Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2“Overview of principles of hybridization and the strategy of nucleicacid probe assays”, Elsevier. N.Y. (1993); and Current Protocols inMolecular Biology, Chapter 2, Ausubel, et al., Eds., Greene Publishingand Wiley-Interscience, New York (1995).

Detection probes are preferably selected to be “substantially”complementary to one of the strands of the double stranded DNA ampliconsgenerated by an amplification reaction in a method of the invention.Preferably the probes are substantially complementary to theimmobilizable (e.g. biotin labelled) antisense strands of the ampliconsgenerated from the target DNA.

It is allowable for detection probes to contain one or more mismatchesto their target sequence. In general, sequences that exhibit at least65%, more preferably at least 80% homology with the target DNAoligonucleotide sequences are considered suitable for use in a method ofthe present invention.

The step of analysing the anucleated blood cell-extracted nucleic acidfraction for the presence of a disease marker can thus be performed bystandard nucleic acid analysis techniques. The step of determiningwhether there is an alteration in the level of said nucleic acid markerin said nucleic acid fraction with respect to an unaffected blood samplewill involve (semi-) quantitative measurements of the amount of diseasemarker in the anucleated blood cells. A much preferred protocol for thedetection of disease-specific markers in the nucleic acids isolated fromanucleated blood cells is therefore quantitative reverse-transcriptionPCR (qRT-PCR) (Freeman et al., BioTechniques 26:112-125 (1999)).

An “unaffected blood sample” as referred to above refers to the level ofthe disease marker in anucleated blood cells of a healthy controlsubject or from the same subject prior to the onset of the disease.Since anucleated blood cell characteristics and quantities of anucleatedblood cell components depend on, amongst other things, species and age,it is preferable that the non-diseased control anucleated blood cellscome from a subject of the same species, age and from the samesub-population (e.g. smoker/nonsmoker). Alternatively, control data maybe taken from databases and literature. It will be appreciated that thecontrol sample may also be taken from the diseased subject at aparticular time-point, in order to analyze the progression of thedisease.

Disease markers include cancer/specific mutations and cancer-specificmutations may include a wide variety of mutations known to be associatedwith cancer. A non-limiting list of examples of mutations for variouscancers is provided at http://www.sanger.ac.uk/genetics/CGP/Census/ andin the Tables herein.

The invention further provides a kit for diagnosing disease in asubject, the kit comprising a packaging material which comprises atleast one agent for specifically determining a level and/or activity ofat least one nucleic acid mutant and/or nucleic acid profile in ananucleated blood cell sample of the subject. As used herein, the term“diagnosing” refers to determining the presence of a disease,classifying a disease, determining a severity of disease (grade orstage), monitoring disease progression, forecasting an outcome of thedisease and/or prospects of recovery.

It will be appreciated that the tools necessary for detecting thedisease-derived nucleic acid may be provided as a kit, such as anFDA-approved kit, which may contain one or more unit dosage formcontaining the active ingredient for detection of the disease-derivednucleic acid in anucleated blood cells by a method of the presentinvention.

Alternatively, the kit may comprise means for collecting the sample andspecific amplification and/or detection primers packaged separately.

The kit may be accompanied by instructions for performing a method ofthe present invention.

For example, the kit may be comprised in a device such as a dipstick ora cartridge, (optionally comprised in a housing) to which a blood sampleor an isolated and/or amplified anucleated blood cell nucleic acidsample may be applied and which detects a disease-derived ordisease-specific nucleic acid or nucleic acid profile in said sample.The device may comprise any agent capable of specifically detecting thedisease-derived nucleic acid. For example, the device may comprise oneor a combination of immobilized mutation-specific hybridization probesthat bind the disease-derived nucleic acid and an indicator fordetecting binding. In an embodiment of this invention, supports areprovided in the device to which the hybridization probes are removablyor fixedly attached.

According to one embodiment, the device may be a lateral flow devicecomprising inlet means for flowing a blood sample or an isolated and/oramplified anucleated blood cell nucleic acid sample into contact withthe agents capable of detecting the disease-derived nucleic acid. Thetest device can also include a flow control means for assuring that thetest is properly operating. Such flow control means can include controlnucleic acids bound to a support which capture detection probes added tothe sample as a means of confirming proper flow of sample fluid throughthe test device. Alternatively, the flow control means can includecapture probes in the control region which capture control nucleic acidsnaturally present in said sample or added thereto as control, againindicating that proper flow is taking place within the device.

In another aspect, the present invention provides the use of device ofthe present invention for diagnosing disease in a subject using any oneof the methods described herein above. Very suitable devices for use indiagnosing disease in a subject using any one of the methods describedherein above include Platelet RNA chips such as for instance describedin Nagalla & Bray (2010) Blood 115 (1): 2-3 and Gnatenko et al. Blood115 (1): 7-14.

The invention will now be exemplified by means of the followingnon-limiting examples.

EXAMPLES Example 1

Thrombocytes were isolated from blood samples of 4 glioblastoma patientsand 4 healthy donors by centrifugation steps. The thrombocytes were thensubjected to RNA extraction using Trizol RNA isolation. The purifiedthrombocytic RNA samples were then converted to cDNA and analyzed byAgilent 4x44K expression microarrays using standard microarrayprotocols. This allowed the profiling of the mRNAs in the differentthrombocyte preparations.

About 8500 RNA transcripts could not be detected by expressionmicroarrays in platelets from healthy donors. These transcripts werepresent at levels below the detection limit of the Agilent 4x44K chip inthrombocytes from healthy donors. Hence, such RNAs may all be potentialbiomarkers for cancer diagnostics. Of the RNAs not detected byexpression microarrays in thrombocytes from healthy donors, asubstantial set of RNAs was detected in thrombocytes from glioblastomapatients. Table 1 summarizes unique thrombocytic RNA transcriptsdetected in thrombocytes from glioblastoma patients but not inthrombocytes from healthy donors by expression microarrays. Unique RNAtranscripts detected in 4/4 patient samples (Table 1A) or in 3/4 patientsamples (Table 1B), but not in any of the four control samples aresummarized in Table 1.

TABLE 1 Unique thrombocytic RNA transcripts detected in thrombocytesfrom glioblastoma patients but not in thrombocytes from healthy donorsby expression microarrays. 1A. Transcripts detected in thrombocytes infour out of four patient samples, but not detected in thrombocytes fromcontrol samples. A_23_P207233 A_23_P47546 A_24_P452024 A_24_P642240A_24_P654255 A_24_P712193 A_24_P816073 A_32_P142521 A_32_P167111A_32_P35839 A_32_P59532 AA594975 AF035790 AF119839 AF130062 AI138440AK098562 ASPM AW269819 BC002534 BC024745 BC047055 BHMT BM683433 BX118161C10orf10 C9orf138 CCL16 CENPQ CLN5 CLTCL1 COCH CPA6 CUTL2 DKFZp547H025DLSTP DNAJC5B ENST00000303697 ENST00000315208 ENST00000382726 FILIP1 GALGPR149 GTSE1 HAS3 HFE HOXB6 HOXD11 IGF1 IL21 LDLR LOC221710 LOC388160LOC641999 LRRC2 LRRC4 MGC16291 MPDZ MYCL1 NPR3 OLAH OR2H1 PLK4 PNMA2ROBO4 SEPT10 SLC14A1 SP2 SPANXB2 TAF5L TCEAL7 THC2279825 THC2334717THC2340924 THC2412206 TIMP4 TMPRSS3 TNFAIP6 TNK1 ZNF596 1B. Transcriptsdetected in thrombocytes in three out of four patient samples, but notdetected in thrombocytes from control samples. A_23_P72252 A_24_P195400A_24_P195621 KRT8P23 A_24_P246777 A_24_P315255 A_24_P647965 A_24_P669822A_24_P752208 A_24_P790361 A_24_P834066 A_24_P915245 A_24_P928453A_24_P929126 A_24_P931713 A_24_P933278 A_24_P934497 A_24_P935492A_32_P119949 A_32_P136427 A_32_P15328 A_32_P182135 A_32_P69993A_32_P743731 A_32_P75311 A_32_P92274 AA420988 AA669267 AA843546 AA890136AA918648 ABCA10 ABCB9 ACADL ACE ADAM32 AF119848 AF136408 AF217973AF263545 AF315716 AF401032 AI291464 AI335947 AI885257 AK021897 AK057725AK057935 AK074369 AK091028 AK096102 AK096991 AK130038 AL133089 ALDH5A1ANKRD40 APOA1 APOA1 APOD AW385956 AY358234 BC017851 BC037882 BC038740BC041899 BC37295_3 BCL2L11 BF376089 BF435769 BF509481 BF826743 BHMT2BHMT2 BM476468 BM681332 BPI BQ028381 BX091616 BX647685 C15orf37 C17orf53C18orf56 C20orf117 C3orf23 C4orf6 C4orf7 C6orf10 C6orf52 C9orf39 CART1CC2D1A CCL7 CDC2 CES4 CF527929 CITED4 CLDN4 CNTN2 COL1A1 COL5A2 COL6A1COX11 CPLX2 CPNE6 CRB1 DB380193 DENND1A DPPA5 DST EFEMP1 EGFRENST00000254271 ENST00000258873 ENST00000272235 ENST00000295989ENST00000299308 ENST00000300996 ENST00000315293 ENST00000335534ENST00000354261 ENST00000354417 ENST00000355077 ENST00000355247ENST00000356104 ENST00000369615 ENST00000374334 ENST00000374458ENST00000375587 ENST00000381050 ENTPD8 EPS8L3 ERVWE1 ESX1 F8 FAM104BFAM62C FAM71B FAM9C FBXW10 FCRL4 FGFR1 FLJ25715 FLJ32312 FLJ37543FLJ39582 FLJ39779 FNDC5 FRG2 FSIP2 FTCD GAPDHS GAS2L2 GAS8 GCKR GLRA1GPR143 GPR98 GUCA1C HILS1 HLA-DRB6 HOXD3 HR44 IGF1 IGF1 IGSF4 INHBAITGAV JPH1 KIAA0492 KIAA1661 KIF20A KLHL9 KREMEN1 LCE3B LENEP LIFRLOC222171 LOC339524 LOC348021 LOC388503 LOC390211 LOC440295 LOC642730LOC643100 LOC643125 LOC648556 LOC92270 M31157 MGC39584 MGC43122 MRAPMSTO1 MYLC2PL MYO7A NDST3 NF2 NNMT NR1H4 NTN1 NTRK3 NTS NXPH3 ODAM OPCMLOR8H1 PALM2-AKAP2 PAX9 PCNXL2 PHC2 PKNOX1 PLAC1 POTE2 PPCDC PPFIBP1PPP1R14C RBMY2EP RCBTB1 RHOD RRAGB RSHL1 RSPO1 S72478 SAA4 SASS6 SDK1SLC22A9 SLC26A9 SLCO1A2 SNAP25 SP5 SPBC25 STEAP1 SUFU SUNC1 SYCE1 SYT12TAS2R38 TAS2R4 TBC1D3 TBC1D8B TCEB3C TEK THC2269604 THC2269920THC2276996 THC2279230 THC2281591 THC2281747 THC2286878 THC2286962THC2289112 THC2296760 THC2316481 THC2316929 THC2339079 THC2339904THC2347643 THC2369034 THC2374304 THC2374505 THC2380237 THC2385918THC2407039 THC2444579 THC2454812 THRSP TM4SF20 TNFRSF13B TREH TRPA1TRPC7 TSC22D2 TSHZ2 TTTY6 UGT8 UNC13B USP2 USP6 VLDLR WWTR1 X87895 ZNF28ZNF57

TABLE 2 Cancer-specific mutations for various cancers. Symbol GeneID ChrBand Tumour Types (Somatic Mutations) Cancer Syndrome ABL1 25 9q34.1CML, ALL, T-ALL ABL2 27 1q24-q25 AML ACSL3 2181 2q36 prostate AF15Q1457082 15q14 AML AF1Q 10962 1q21 ALL AF3p21 51517 3p21 ALL AF5q31 271255q31 ALL AKAP9 10142 7q21-q22 papillary thyroid AKT1 207 14q32.32breast, colorectal, ovarian, NSCLC AKT2 208 19q13.1-q13.2 ovarian,pancreatic ALK 238 2p23 ALCL, NSCLC, Neuroblastoma Familialneuroblastoma ALO17 57714 17q25.3 ALCL APC 324 5q21 colorectal,pancreatic, desmoid, Adenomatous polyposis coli; hepatoblastoma, glioma,other CNS Turcot syndrome ARHGEF12 23365 11q23.3 AML ARHH 399 4p13 NHLARNT 405 1q21 AML ASPSCR1 79058 17q25 alveolar soft part sarcoma ASXL1171023 20q11.1 MDS, CMML ATF1 466 12q13 malignant melanoma of softparts, angiomatoid fibrous histiocytoma ATIC 471 2q35 ALCL ATM 47211q22.3 T-PLL, leukemia, lymphoma, Ataxia-telangiectasiamedulloblastoma, glioma BCL10 8915 1p22 MALT BCL11A 53335 2p13 B-CLLBCL11B 64919 14q32.1 T-ALL BCL2 596 18q21.3 NHL, CLL BCL3 602 19q13 CLLBCL5 603 17q22 CLL BCL6 604 3q27 NHL, CLL BCL7A 605 12q24.1 BNHL BCL9607 1q21 B-ALL BCR 613 22q11.21 CML, ALL, AML BHD 201163 17p11.2 renal,fibrofolliculomas, trichodiscomas Birt-Hogg-Dube syndrome BIRC3 33011q22-q23 MALT BLM 641 15q26.1 leukemia, lymphoma, skin squamous BloomSyndrome cell, other cancers BMPR1A 657 10q22.3 gastrointestinal polypsJuvenile polyposis BRAF 673 7q34 melanoma, colorectal, papillarythyroid, borderline ov, Non small-cell lung cancer (NSCLC),cholangiocarcinoma, pilocytic astrocytoma BRCA1 672 17q21 ovarian,breast, Hereditary breast/ovarian cancer BRCA2 675 13q12 breast,ovarian, pancreatic, leukemia Hereditary breast/ovarian (FANCB, FANCD1)cancer BRD3 8019 9q34 lethal midline carcinoma of young people BRD423476 19p13.1 lethal midline carcinoma of young people BRIP1 83990 17q22AML, leukemia, breast Fanconi anaemia J, breast cancer susceptiblityBTG1 694 12q22 BCLL BUB1B 701 15q15 rhabdomyosarcoma Mosaic variegatedaneuploidy C12orf9 93669 12q14.3 lipoma C15orf21 283651 15q21.1 prostateCANT1 124583 17q25 prostate CARD11 84433 7p22 DLBL CARS 833 11p15.5 ALCLCBFA2T1 862 8q22 AML CBFA2T3 863 16q24 AML CBFB 865 16q22 AML CBL 86711q23.3 AML, JMML, MDS CBLB 868 3q13.11 AML CBLC 23624 19q13.2 AML CCND1595 11q13 CLL, B-ALL, breast CCND2 894 12p13 NHL, CLL CCND3 896 6p21 MMCD74 972 5q32 NSCLC CD79A 973 19q13.2 DLBCL CD79B 974 17q23 DLBCL CDH1999 16q22.1 lobular breast, gastric Familial gastric carcinoma CDH111009 16q22.1 aneurysmal bone cysts CDK4 1019 12q14 melanoma Familialmalignant melanoma CDK6 1021 7q21-q22 ALL CDKN2A- 1029 9p21 melanoma,multiple other tumour types, Familial malignant melanoma p16(INK4a)pancreatic CDKN2A- 1029 9p21 melanoma, multiple other tumour types,Familial malignant melanoma p14ARF pancreatic CDKN2C 1031 1p32 glioma,MM CDX2 1045 13q12.3 AML CEBPA 1050 19q13.1 AML, MDS CEP1 11064 9q33MPD, NHL CHCHD7 79145 8q11.2 salivary adenoma CHEK2 11200 22q12.1 breastfamilial breast cancer CHIC2 26511 4q11-q12 AML CHN1 1123 2q31-q32.1extraskeletal myxoid chondrosarcoma CIC 23152 19q13.2 soft tissuesarcoma CLTC 1213 17q11-qter ALCL, renal CLTCL1 8218 22q11.21 ALCLCMKOR1 57007 2q37.3 lipoma COL1A1 1277 17q21.31-q22 dermatofibrosarcomaprotuberans, aneurysmal bone cyst COPEB 1316 10p15 prostate, gliomaCOX6C 1345 8q22-q23 uterine leiomyoma CREB1 1385 2q34 clear cellsarcoma, angiomatoid fibrous histiocytoma CREB3L2 64764 7q34 fibromyxoidsarcoma CREBBP 1387 16p13.3 AL, AML CRLF2 64109 Xp22.3; Yp11.3 B-ALL,Downs associated ALL CRTC3 64784 15q26.1 salivary gland mucoepidermoidCTNNB1 1499 3p22-p21.3 colorectal, cvarian, hepatoblastoma, others,pleomorphic salivary adenoma CYLD 1540 16q12-q13 cylindroma Familialcylindromatosis D10S170 8030 10q21 papillary thyroid, CML DDB2 164311p12 skin basal cell, skin squamous cell, Xeroderma pigmentosum (E)melanoma DDIT3 1649 12q13.1-q13.2 liposarcoma DDX10 1662 11q22-q23 AML*DDX5 1655 17q21 prostate DDX6 1656 11q23.3 B-NHL DEK 7913 6p23 AMLDICER1 23405 14q32.13 pleuropulmonary blastoma Familial PleuropulmonaryBlastoma DUX4 22947 4q35 soft tissue sarcoma EGFR 1956 7p12.3-p12.1glioma, NSCLC Familial lung cancer EIF4A2 1974 3q27.3 NHL ELF4 2000 Xq26AML ELK4 2005 1q32 prostate ELKS 23085 12p13.3 papillary thyroid ELL8178 19p13.1 AL ELN 2006 7q11.23 B-ALL EML4 27436 2p21 NSCLC EP300 203322q13 colorectal, breast, pancreatic, AML EPS15 2060 1p32 ALL ERBB2 206417q21.1 breast, ovarian, other tumour types, NSCLC, gastric ERCC2 206819q13.2-q13.3 skin basal cell, skin squamous cell, Xeroderma pigmentosum(D) melanoma ERCC3 2071 2q21 skin basal cell, skin squamous cell,Xeroderma pigmentosum (B) melanoma ERCC4 2072 16p13.3-p13.13 skin basalcell, skin squamous cell, Xeroderma pigmentosum (F) melanoma ERCC5 207313q33 skin basal cell, skin squamous cell, Xeroderma pigmentosum (G)melanoma ERG 2078 21q22.3 Ewing sarcoma, prostate, AML ETV1 2115 7p22Ewing sarcoma, prostate ETV4 2118 17q21 Ewing sarcoma, Prostatecarcinoma ETV5 2119 3q28 Prostate ETV6 2120 12p13 congenitalfibrosarcoma, multiple leukemia and lymphoma, secretory breast, MDS, ALLEVI1 2122 3q26 AML, CML EWSR1 2130 22q12 Ewing sarcoma, desmoplasticsmall round cell tumor, ALL, clear cell sarcoma, sarcoma, myoepitheliomaEXT1 2131 8q24.11-q24.13 exostoses, osteosarcoma Multiple Exostoses Type1 EXT2 2132 11p12-p11 exostoses, osteosarcoma Multiple Exostoses Type 2EZH2 2146 7q35-q36 DLBCL FACL6 23305 5q31 AML, AEL FANCA 2175 16q24.3AML, leukemia Fanconi anaemia A FANCC 2176 9q22.3 AML, leukemia Fanconianaemia C FANCD2 2177 3p26 AML, leukemia Fanconi anaemia D2 FANCE 21786p21-p22 AML, leukemia Fanconi anaemia E FANCF 2188 11p15 AML, leukemiaFanconi anaemia F FANCG 2189 9p13 AML, leukemia Fanconi anaemia G FBXW755294 4q31.3 colorectal, endometrial, T-ALL FCGR2B 2213 1q23 ALL FEV54738 2q36 Ewing sarcoma FGFR1 2260 8p11.2-p11.1 MPD, NHL FGFR1OP 111166q27 MPD, NHL FGFR2 2263 10q26 gastric. NSCLC, endometrial FGFR3 22614p16.3 bladder, MM, T-cell lymphoma FH 2271 1q42.1 lieomyomatosis, renalhereditary leiomyomatosis and renal cell cancer FIP1L1 81608 4q12idiopathic hypereosinophilic syndrome FLI1 2313 11q24 Ewing sarcoma FLT32322 13q12 AML, ALL FNBP1 23048 9q23 AML FOXL2 668 3q23 granulosa-celltumour of the ovary FOXO1A 2308 13q14.1 alveolar rhabdomyosarcomasFOXO3A 2309 6q21 AL FOXP1 27086 3p14.1 ALL FSTL3 10272 19p13 B-CLL FUS2521 16p11.2 liposarcoma, AML, Ewing sarcoma, angiomatoid fibroushistiocytoma, fibromyxoid sarcoma FVT1 2531 18q21.3 B-NHL GAS7 8522 17pAML* GATA1 2623 Xp11.23 megakaryoblastic leukemia of Downs SyndromeGATA2 2624 3q21.3 AML(CML blast transformation) GATA3 2625 10p15 breastGMPS 8833 3q24 AML GNAQ 2776 9q21 uveal melanoma GNAS 2778 20q13.2pituitary adenoma GOLGA5 9950 14q papillary thyroid GOPC 57120 6q21glioblastoma GPC3 2719 Xq26.1 Wilms tumour Simpson-Golabi-Behmelsyndrome GPHN 10243 14q24 AL GRAF 23092 5q31 AML, MDS HCMOGT-1 9252117p11.2 JMML HEAB 10978 11q12 AML HEI10 57820 14q11.1 uterine leiomyomaHERPUD1 9709 16q12.2-q13 prostate HIP1 3092 7q11.23 CMML HIST1H4I 82946p21.3 NHL HLF 3131 17q22 ALL HLXB9 3110 7q36 AML HMGA1 3159 6p21microfollicular thyroid adenoma, various benign mesenchymal tumors HMGA28091 12q15 lipoma HNRNPA2B1 3181 7p15 prostate HOOK3 84376 8p11.21papillary thyroid HOXA11 3207 7p15-p14.2 CML HOXA13 3209 7p15-p14.2 AMLHOXA9 3205 7p15-p14.2 AML* HOXC11 3227 12q13.3 AML HOXC13 3229 12q13.3AML HOXD11 3237 2q31-q32 AML HOXD13 3239 2q31-q32 AML* HRAS 3265 11p15.5infrequent sarcomas, rare other types, Costello syndromerhadomyosarcoma, ganglioneuroblastoma, bladder HRPT2 3279 1q21-q31parathyroid adenoma, mulitiple Hyperparathyroidism-jaw ossifying jawfibroma tumor syndrome HSPCA 3320 14q32.31 NHL HSPCB 3326 6p12 NHL IDH13417 2q33.3 gliobastoma IDH2 3418 15q26.1 GBM IGH@ 3492 14q32.33 MM,Burkitt lymphoma, NHL, CLL, B-ALL, MALT, MLCLS IGK@ 50802 2p12 Burkittlymphoma, B-NHL IGL@ 3535 22q11.1-q11.2 Burkitt lymphoma IKZF1 103207p12.2 ALL IL2 3558 4q26-q27 intestinal T-cell lymphoma IL21R 5061516p11 NHL IL6ST 3572 5q11 hepatocellular ca IRF4 3662 6p25-p23 MM IRTA183417 1q21 B-NHL ITK 3702 5q31-q32 peripheral T-cell lymphoma JAK1 37161p32.3-p31.3 ALL JAK2 3717 9p24 ALL, AML, MPD, CML JAK3 3718 19p13.1acute megakaryocytic leukemia, JAZF1 221895 7p15.2-p15.1 endometrialstromal tumours JUN 3725 1p32-p31 sarcoma KDM5A 5927 12p11 AML KDM5C8242 Xp11.22-p11.21 clear cell renal carcinoma KDM6A 7403 Xp11.2 renal,oesophageal SCC, MM KDR 3791 4q11-q12 NSCLC, angiosarcoma KIAA1549 576707q34 pilocytic astrocytoma KIT 3815 4q12 GIST, AML, TGCT, mastocytosis,Familial gastrointestinal mucosal melanoma, epithelioma stromal tumourKLK2 3817 19q13.41 prostate KRAS 3845 12p12.1 pancreatic, colorectal,lung, thyroid, AML, others KTN1 3895 14q22.1 papillary thryoid LAF4 38992q11.2-q12 ALL, T-ALL LASP1 3927 17q11-q21.3 AML LCK 3932 1p35-p34.3T-ALL LCP1 3936 13q14.1-q14.3 NHL LCX 80312 10q21 AML LHFP 10186 13q12lipoma LIFR 3977 5p13-p12 salivary adenoma LMO1 4004 11p15 T-ALL LMO24005 11p13 T-ALL LPP 4026 3q28 lipoma, leukemia LYL1 4066 19p13.2-p13.1T-ALL MADH4 4089 18q21.1 colorectal, pancreatic, small intestine,Juvenile polyposis gastrointestinal polyps MAF 4094 16q22-q23 MM MAFB9935 20q11.2-q13.1 MM MALT1 10892 18q21 MALT MAML2 84441 11q22-q23salivary gland mucoepidermoid MAP2K4 6416 17p11.2 pancreatic, breast,colorectal MDM2 4193 12q15 sarcoma, glioma, colorectal, other MDM4 41941q32 GBM, bladder, retinoblastoma MDS1 4197 3q26 MDS, AML MDS2 2592831p36 MDS MECT1 94159 19p13 salivary gland mucoepidermoid MEN1 4221 11q13parathyroid tumors, parathyroid Multiple Endocrine Neoplasia adenoma,pituitary adenoma, pancreatic Type 1 islet cell, carcinoid MET 4233 7q31papillary renal, head-neck squamous cell Familial Papillary Renal CancerMHC2TA 4261 16p13 NHL MITF 4286 3p14.1 melanoma MKL1 57591 22q13 acutemegakaryocytic leukemia MLF1 4291 3q25.1 AML MLH1 4292 3p21.3colorectal, endometrial, ovarian, CNS Hereditary non-polyposiscolorectal cancer, Turcot syndrome MLL 4297 11q23 AML, ALL MLLT1 429819p13.3 AL MLLT10 8028 10p12 AL MLLT2 4299 4q21 AL MLLT3 4300 9p22 ALLMLLT4 4301 6q27 AL MLLT6 4302 17q21 AL MLLT7 4303 Xq13.1 AL MN1 433022q13 AML, meningioma MPL 4352 p34 MPD Familial essentialthrombocythemia MSF 10801 17q25 AML* MSH2 4436 2p22-p21 colorectal,endometrial, ovarian Hereditary non-polyposis colorectal cancer MSH62956 2p16 colorectal, endometrial, ovarian Hereditary non-polyposiscolorectal cancer MSI2 124540 17q23.2 CML MSN 4478 Xq11.2-q12 ALCL MTCP14515 Xq28 T cell prolymphocytic leukemia MUC1 4582 1q21 B-NHL MUTYH 45951p34.3-1p32.1 colorectal Adenomatous polyposis coli MYB 4602 6q22-23adenoid cystic carcinoma MYC 4609 8q24.12-q24.13 Burkitt lymphoma,amplified in other cancers, B-CLL MYCL1 4610 1p34.3 small cell lung MYCN4613 2p24.1 neuroblastoma MYH11 4629 16p13.13-p13.12 AML MYH9 462722q13.1 ALCL MYST4 23522 10q22 AML NACA 4666 12q23-q24.1 NHL NBS1 46838q21 NHL, glioma, medulloblastoma, Nijmegen breakage syndromerhabdomyosarcoma NCOA1 8648 2p23 alveolar rhadomyosarcoma NCOA2 104998q13.1 AML NCOA4 8031 10q11.2 papillary thyroid NF1 4763 17q12neurofibroma, glioma Neurofibromatosis type 1 NF2 4771 22q12.2meningioma, acoustic neuroma, renal Neurofibromatosis type 2 NFIB 47819p24.1 adenoid cystic carcinoma, lipoma NFKB2 4791 10q24 B-NHL NIN 5119914q24 MPD NONO 4841 Xq13.1 papillary renal cancer NOTCH1 4851 9q34.3T-ALL NOTCH2 4853 1p13-p11 marginal zone lymphoma, DLBCL NPM1 4869 5q35NHL, APL, AML NR4A3 8013 9q22 extraskeletal myxoid chondrosarcoma NRAS4893 1p13.2 melanoma, MM, AML, thyroid NSD1 64324 5q35 AML NTRK1 49141q21-q22 papillary thyroid NTRK3 4916 15q25 congenital fibrosarcoma,Secretory breast NUMA1 4926 11q13 APL NUP214 8021 9q34.1 AML, T-ALLNUP98 4928 11p15 AML NUT 256646 q13 lethal midline carcinoma of youngpeople OLIG2 10215 21q22.11 T-ALL OMD 4958 9q22.31 aneurysmal bone cystsP2RY8 286530 Xp22.3; Yp11.3 B-ALL, Downs associated ALL PAFAH1B2 504911q23 MLCLS PALB2 79728 16p12.1 Wilms tumor, medulloblastoma, AML,Fanconi anaemia N, breast breast cancer susceptibility PAX3 5077 2q35alveolar rhabdomyosarcoma PAX5 5079 9p13 NHL, ALL, B-ALL PAX7 50811p36.2-p36.12 alveolar rhabdomyosarcoma PAX8 7849 2q12-q14 follicularthyroid PBX1 5087 1q23 pre B-ALL, myoepithelioma PCM1 5108 8p22-p21.3papillary thyroid, CML, MPD PCSK7 9159 11q23.3 MLCLS PDE4DIP 9659 1q12MPD PDGFB 5155 22q12.3-q13.1 DFSP PDGFRA 5156 4q11-q13 GIST, idiopathichypereosinophilic syndrome PDGFRB 5159 5q31-q32 MPD, AML, CMML, CML PER15187 17p13.1-17p12 AML, CMML PHOX2B 8929 4p12 neuroblastoma familialneuroblastoma PICALM 8301 11q14 TALL, AML, PIK3CA 5290 3q26.3colorectal, gastric, gliobastoma, breast PIK3R1 5295 5q13.1 gliobastoma,ovarian, colorectal PIM1 5292 6p21.2 NHL PLAG1 5324 8q12 salivaryadenoma PML 5371 15q22 APL, ALL PMS1 5378 2q31-q33 colorectal,endometrial, ovarian Hereditary non-polyposis colorectal cancer PMS25395 7p22 colorectal, endometrial, ovarian, Hereditary non-polyposismedulloblastoma, glioma colorectal cancer, Turcot syndrome PMX1 53961q24 AML PNUTL1 5413 22q11.2 AML POU2AF1 5450 11q23.1 NHL POU5F1 54606p21.31 sarcoma PPARG 5468 3p25 follicular thyroid PRCC 5546 1q21.1papillary renal PRDM16 63976 1p36.23-p33 MDS, AML PRF1 5551 10q22various leukaemia, lymphoma PRKAR1A 5573 17q23-q24 myxoma, endocrine,papillary thyroid Carney complex PRO1073 29005 11q31.1 renal cellcarcinoma (childhood epithelioid) PSIP2 11168 9p22.2 AML PTCH 57279q22.3 skin basal cell, medulloblastoma Nevoid Basal Cell CarcinomaSyndrome PTEN 5728 10q23.3 harmartoma, glioma, prostate, CowdenSyndrome, Bannayan- endometrial Riley-Ruvalcaba syndrome PTPN11 578112q24.1 JMML, AML, MDS RAB5EP 9135 17p13 CMML RAD51L1 5890 14q23-q24.2lipoma, uterine leiomyoma RAF1 5894 3p25 pilocytic astrocytoma RANBP1764901 5q34 ALL RAP1GDS1 5910 4q21-q25 T-ALL RARA 5914 17q12 APL RB1 592513q14 retinoblastoma, sarcoma, breast, small Familial retinoblastomacell lung RBM15 64783 1p13 acute megakaryocytic leukemia RECQL4 94018q24.3 osteosarcoma, skin basal and sqamous Rothmund-Thompson Syndromecell REL 5966 2p13-p12 Hodgkin Lymphoma RET 5979 10q11.2 medullarythyroid, papillary thyroid, Multiple endocrine neoplasiapheochromocytoma 2A/2B ROS1 6098 6q22 glioblastoma, NSCLC RPL22 61461p36.31 AML, CML RPN1 6184 3q21.3-q25.2 AML RUNX1 861 21q22.3 AML, preB-ALL, T-ALL RUNXBP2 7994 8p11 AML SBDS 51119 7q11 AML, MDSSchwachman-Diamond syndrome SDH5 54949 11q12.2 paraganglioma Familialparaganglioma SDHB 6390 1p36.1-p35 paraganglioma, pheochromocytomaFamilial paraganglioma SDHC 6391 1q21 paraganglioma, pheochromocytomaFamilial paraganglioma SDHD 6392 11q23 paraganglioma, pheochromocytomaFamilial paraganglioma SEPT6 23157 Xq24 AML SET 6418 9q34 AML SETD229072 3p21.31 clear cell renal carcinoma SFPQ 6421 1p34.3 papillaryrenal cell SFRS3 6428 6p21 follicular lymphoma SH3GL1 6455 19p13.3 ALSIL 6491 1p32 T-ALL SLC45A3 85414 1q32 prostate SMARCA4 6597 19p13.2NSCLC SMARCB1 6598 22q11 malignant rhabdoid Rhabdoid predispositionsyndrome SMO 6608 7q31-q32 skin basal cell SOCS1 8651 16p13.13 HodgkinLymphoma, PMBL SRGAP3 9901 3p25.3 pilocytic astrocytoma SS18 676018q11.2 synovial sarcoma SS18L1 26039 20q13.3 synovial sarcoma SSH3BP110006 10p11.2 AML SSX1 6756 Xp11.23-p11.22 synovial sarcoma SSX2 6757Xp11.23-p11.22 synovial sarcoma SSX4 6759 Xp11.23 synovial sarcoma STK116794 19p13.3 NSCLC, pancreatic, jejunal Peutz-Jeghers syndromeharmartoma, ovarian, testicular STL 7955 6q23 B-ALL SUFU 51684 10q24.32medulloblastoma Medulloblastoma predisposition SUZ12 23512 17q11.2endometrial stromal tumours SYK 6850 9q22 MDS, peripheral T-celllymphoma TAF15 8148 17q11.1-q11.2 extraskeletal myxoid chondrosarcomas,ALL TAL1 6886 1p32 lymphoblastic leukemia/biphasic TAL2 6887 9q31 T-ALLTCEA1 6917 8q11.2 salivary adenoma TCF1 6927 12q24.2 hepatic adenoma,hepatocellular ca Familial Hepatic Adenoma TCF12 6938 15q21extraskeletal myxoid chondrosarcoma TCF3 6929 19p13.3 pre B-ALL TCL1A8115 14q32.1 T-CLL TCL6 27004 14q32.1 T-ALL TET2 54790 4q24 MDS TFE37030 Xp11.22 papillary renal, alveolar soft part sarcoma, renal TFEB7942 6p21 renal (childhood epithelioid) TFG 10342 3q11-q12 papillarythyroid, ALCL, NSCLC TFPT 29844 19q13 pre-B ALL TFRC 7037 3q29 NHLTHRAP3 9967 1p34.3 aneurysmal bone cysts TIF1 8805 7q32-q34 APL TLX13195 10q24 T-ALL TLX3 30012 5q35.1 T-ALL TMPRSS2 7113 21q22.3 prostateTNFAIP3 7128 6q23 marginal zone B-cell lymphomas, Hodgkin's lymphoma,primary mediastinal B cell lymphoma TNFRSF17 608 16p13.1 intestinalT-cell lymphoma TNFRSF6 355 10q24.1 TGCT, nasal NK/T lymphoma, skinsquamous cell ca -burn scar-related TOP1 7150 20q12-q13.1 AML* TP53 715717p13 breast, colorectal, lung, sarcoma, Li-Fraumeni syndromeadrenocortical, glioma, multiple other tumour types TPM3 7170 1q22-q23papillary thyroid, ALCL TPM4 7171 19p13.1 ALCL TPR 7175 1q25 papillarythyroid TRA@ 6955 14q11.2 T-ALL TRB@ 6957 7q35 T-ALL TRD@ 6964 14q11T-cell leukemia TRIM27 5987 6p22 papillary thyroid TRIM33 51592 1p13papillary thyroid TRIP11 9321 14q31-q32 AML TSC1 7248 9q34 hamartoma,renal cell Tuberous sclerosis 1 TSC2 7249 16p13.3 hamartoma, renal cellTuberous sclerosis 2 TSHR 7253 14q31 toxic thyroid adenoma TTL 1504652q13 ALL USP6 9098 17p13 aneurysmal bone cysts VHL 7428 3p25 renal,hemangioma, pheochromocytoma von Hippel-Lindau syndrome WAS 7454Xp11.23-p11.22 lymphoma Wiskott-Aldrich syndrome WHSC1 7468 4p16.3 MMWHSC1L1 54904 8p12 AML WRN 7486 8p12-p11.2 osteosarcoma, meningioma,others Werner Syndrome WT1 7490 11p13 Wilms, desmoplastic small roundcell Denys-Drash syndrome, Frasier tumor syndrome, Familial Wilms tumorWTX 139285 Xq11.1 Wilms tumour XPA 7507 9q22.3 skin basal cell, skinsquamous cell, Xeroderma pigmentosum (A) melanoma XPC 7508 3p25 skinbasal cell, skin squamous cell, Xeroderma pigmentosum (C) melanomaZNF145 7704 11q23.1 APL ZNF198 7750 13q11-q12 MPD, NHL ZNF278 2359822q12-q14 Ewing sarcoma ZNF331 55422 19q13.3-q13.4 follicular thyroidadenoma ZNF384 171017 12p13 ALL ZNF521 25925 18q11.2 ALL ZNF9 7555 3q21aneurysmal bone cysts ZNFN1A1 10320 7p12 ALL, DLBL

Example 2 Introduction

Diagnostic platforms which are highly predictive for diagnosing,monitoring, and stratifying cancer patients are key instruments in thedevelopment of personalized medicine. In this Example, it isdemonstrated that tumor cells transfer (mutant) RNA into blood plateletsin vitro, and it is shown that blood platelets isolated fromglioblastoma and prostate cancer patients contain the cancer-associatedRNA biomarkers EGFRvIII, and PCA3 and PSA, respectively. Moreover, geneexpression arrays revealed a distinct mRNA signature in platelets fromglioma patients as compared to normal control subjects. Becauseplatelets are easily accessible and isolated, they may form anattractive platform for the companion diagnostics of cancer.

Methods Platelet Isolation and Tissue Resection.

Platelets were isolated from whole blood collected in purple-cap BDVacutainers containing EDTA anti-coagulant by standard centrifugation,and quality (activation and aggregation) as well as purity was assessedby microscopic analysis showing less than 0.1% contamination with red orwhite blood cells. Next, isolated platelet pellets were snap-frozen forfurther use. Glioma tissue resection and whole blood harvesting fromglioma and prostate cancer patients was performed at the VU Universitymedical center and Umeå University, as described elsewhere (J. Skog etal., Nat Cell Biol. 10(12), 1470-6 (2008)).

Microvesicle Isolation, Labeling, and Transfer.

Microvesicles were isolated from U87-EGFRvIII glioma cells and labeledas described previously (J. Skog et al., Nat Cell Biol. 10(12), 1470-6(2008)). After U87-dEGFR microvesicle incubation the platelets werewashed and treated with RNAse enzymes to ensure the EGFRvIII RNA wasdelivered into the platelets and therefore protected from RNAse-mediateddegradation. For confocal microscopy analysis the platelets were stainedwith texas red-conjugated wheat germ agglutinin to indicate plateletstructure and analyzed for microvesicle uptake by the presence of greenPKH67. RNA purification. RNA was isolated using miRvana (Ambion) ormiRNeasy (Qiagen) protocols according the manufacturer's instruction.RNA concentration and quality was determined using a Bioanalyzer 2100with total RNA Pico chip (Agilent).

RT-PCR.

RT-PCR for EGFRvIII, PCA3, PSA, and GAPDH, was performed as describedpreviously (J. Skog et al., Nat Cell Biol. 10(12), 1470-6 (2008)) usingthe following primer sets:

GAPDH primers: forward (SEQ ID NO.: 1 5′-GAAGGTGAAGGTCGGAGTC-3′,reverse: (SEQ ID NO: 2) 5′-TCAGAAGATGGTGATGGGATTTC-3′. PSA primers:forward (SEQ ID NO: 3) 5′-ATGTGGGTCCCGGTTGTCTT-3′, reverse(SEQ ID NO: 4) 5′-TCCCACAATCCGAGACAGGA-3′ Nested PCA3 primers: PCR1;forward (SEQ ID NO: 5) 5′-AGTCCGCTGTGAGTCT-3′, reverse (SEQ ID NO: 6)5′-CCATTTCAGCAGATGTGTGG-3′; PCR2: forward (SEQ ID NO: 7)5′-ATCGACGGCACTTTCTGAGT-3′, reverse (SEQ ID NO: 8)5′-TGTGTGGCCTCAGATGGTAA-3′. Nested EGFRvIII primers; PCR1; forward(SEQ ID NO: 9) 5′-CCAGTATTGATCGGGAGAGC-3′, reverse (SEQ ID NO: 10)5′-TGTGGATCCAGAGGAGGAGT-3′; PCR2: forward (SEQ ID NO: 11)5′-GAGCTCTTCGGGGAGCAG-3′, reverse (SEQ ID NO: 12)5′-GCCCTTCGCACTTCTTACAC-3′.

Gene Expression Arrays.

The mRNA expression arrays were performed at the VU University MedicalCenter microarray core facility using Agilent 4x44K gene expressionarrays. Platelet RNA integrity was assessed using the Agilent 2100Bioanalyzer (Agilent Technologies, Inc.). RNA samples were labelledusing the Agilent Low RNA Input Linear Amplification Kit Plus(5188-5340) according to the manufacturer's protocol.

Briefly, 25 ng of total RNA was amplified and reverse transcribed tocDNA using T7-polymerase and subsequently labelled with Cy3 or Cy5. Dyeincorporation was measured using a Nanodrop ND-1000 spectrophotometer.Subsequently, cRNA was hybridized using the Agilent Gene ExpressionHybridization Kit (5188-5242), according to the manufacturer's protocol.Briefly, 825 ng of Cy3 labelled cRNA was mixed with 825 ng of Cy5labelled cRNA, fragmented for 30 min at 60° C. in the dark andhybridized on an Agilent Hybridization Chamber Gasket Slide(G2534-60011) in a rotation oven at 65° C. for 17 h. Slides scannedusing an Agilent Microarray Scanner (G2565BA). Image analysis and arraynormalization was performed using feature extraction software version9.5 (Agilent Technologies, Inc.). The Agilent GE2-v5_95 protocol wasapplied using default settings.

Statistical Analysis.

The heat map (FIG. 3C) of the gene expression data was generated usingmedian centered arrays in Excel (Microsoft Office 2007 package) with theS.A.M. analysis plug-in, with a set false discovery rate <0.5%. Thetop-30 significantly differentially expressed genes are depicted usingHeatmap Builder v1.1 software (King et al. Physiol Genomics. Sep. 21,2005; 23(1):103-118).).

Results

In this Example it is shown that platelets isolated from healthy humancontrol subjects have the ability to take up RNA-containingmicrovesicles derived from human brain tumor cells (glioma), and containtumor-associated RNA, including mutant EGFRvIII. Uptake of PKH67labelled glioma-derived microvesicles is demonstrated in blood plateletsby FACS analysis and confocal microscopy. In addition, it was shown thatmicrovesicle-mediated transfer of mutant EGFRvIII RNA into plateletsfrom healthy control subjects by RT-PCR occurs. Furthermore, it isdetermined that circulating platelets isolated from glioma patients,contain RNA biomarkers (see FIG. 3B). RT-PCR was used to determinewhether mutant EGFRvIII mRNA was found in resected high-grade gliomatissues (n=18) and the result was compared to platelets from the samepatient and to platelets from healthy control subjects (n=30). Thesamples were coded and RT-PCR was performed in a blind assay. Four ofthe 18 (22.5%) glioma samples contained the EGFRvIII transcript, asobserved before. Notably, EGFRvIII could be amplified from platelets in3 out of these 4 EGFRvIII-positive patients (75%), and in none of theplatelets of the healthy donors (n=12), whereas GAPDH mRNA was detectedin all platelet samples. A possible false negative signal was detectedin the platelets of one patient only, which may be contributed toinadequate processing of the blood sample. Conversely, one patient withEGFRvIII-negative tissue sample was EGFRvIII-positive in the plateletsample, most likely due to heterogeneous distribution of EGFRvIIIpositive foci in high-grade gliomas.

To demonstrate that the presence of tumor-associated messages is notunique to platelets from glioma patients we report the presence of mRNAscoding for the prostate cancer markers PCA3 and PSA in platelets fromprostate cancer patients (n=12) and their absence in platelets fromhealthy control subjects (n=10) (See FIG. 4). Finally, using geneexpression arrays it was determined the mRNA expression profiles ofplatelets isolated from healthy control subjects (n=12), and gliomapatients (n=8). Distinct mRNA expression profiles were obtained and aminimal glioma biomarker signature was detected (FIG. 3C, left panel).Interestingly, several of the potential biomarkers were barelydetectable in control samples, whereas in the glioma samples they werehighly expressed (FIG. 3C, right panel).

In conclusion, the findings of the present inventors demonstrate thatblood platelets contain cancer markers in the form of tumor-derived ortumor-associated RNA and, therefore, may serve as a diagnostic platformfor the molecular profiling of cancer in the context of personalizedmedicine.

1. A method of analysing a blood sample of a subject for the presence ofa disease cancer marker, said method comprising the steps of a)extracting nucleic acid from anucleated blood cells, in said bloodsample to provide an anucleated blood cell-extracted nucleic acidfraction, and b) analysing said anucleated blood cell-extracted nucleicacid fraction for the presence of a cancer marker, wherein said cancermarker is a cancer-specific mutation in a gene of a nucleated cell ofsaid subject, or wherein said cancer marker is a cancer-specificexpression profile of genes of a nucleated cell of said subject, whereinsaid cancer marker is not a megakaryocyte derived nucleic acid, andwherein said nucleated cell is not a megakaryocyte.
 2. The method ofclaim 1, wherein said anucleated blood cells are thrombocytes. 3-8.(canceled)
 9. The method of any claim 1, wherein said cancer-specificmutation is in a chromosomal gene, or wherein said cancer-specificexpression profile is of chromosomal genes.
 10. The method of claim 1,wherein said nucleic acid is ribonucleic acid (RNA).
 11. The method ofclaim 1, wherein said step b) of analysing said anucleated bloodcell-extracted nucleic acid fraction for the presence of a cancer markercomprises the selective amplification of i) said mutation by reversetranscriptase polymerase chain reaction amplification using at least onenucleic acid mutation-specific amplification primer or probe, or ii) aplurality of mRNAs by reverse transcriptase polymerase chain reactionamplification to determine the expression level of the chromosomal genesencoding said mRNAs to thereby provide an expression profile for saidgenes and comparing said expression profile to a reference profile. 12.The method of claim 1, wherein said method is part of a method ofdiagnosing said cancer in a subject, and wherein the presence of saidcancer marker in said anucleated blood cell-extracted nucleic acidfraction is indicative of said subject suffering from said cancer.
 13. Amethod for determining the stage of disease or the efficacy of a diseasetreatment in a subject, comprising the steps of: analysing a bloodsample of a subject for the presence of a cancer marker using the methodaccording to claim 1 at a first time point to thereby provide a firstvalue for the level of said marker in said subject, analysing a bloodsample of said subject for the presence of a cancer marker using themethod according to claim 1 at a second time point to thereby provide asecond value for the level of said disease marker in said subject,wherein said subject has been subjected to a treatment between saidfirst and second time point, and comparing said first and second valueto determine the efficacy of said treatment in said subject.
 14. Amethod for determining the stage of a disease in a subject, comprisingthe steps of: analysing a blood sample of a subject for the presence ofa cancer marker using the method according to claim 1 to thereby providea test value for the level of said marker in said subject, providing areference value for the level of said cancer marker wherein saidreference value is correlated to a particular stage of disease, andcomparing said test and reference value to determine the cancer stage insaid subject.
 15. A kit of parts adapted for performing the methodrecited in claim 1, the kit comprising a packaging material whichcomprises at least one of: a container for holding anucleated bloodcells separated from a blood sample of a subject; an agent forextracting nucleic acids from said anucleated blood cells; an agent forselectively amplifying from said nucleic acids extracted from saidanucleated blood cells a disease-specific mutation in a gene of anucleated cell of said subject by reverse transcriptase polymerase chainreaction amplification, and a printed or electronic instruction forperforming the method recited in claim 1, the kit further comprising: areference for said cancer marker, wherein said reference is indicativefor the presence or absence of said marker in said anucleated bloodcells-extracted nucleic acid fraction.
 16. The kit of claim 15, whereinsaid reference is a reference value for the level of nucleic acidscomprising said cancer-specific mutation in thrombocytes in a healthycontrol subject or in a control subject suffering from said cancer, orwherein said reference is a reference expression profile for saidplurality of mRNAs in anucleated blood cells from a healthy controlsubject or from a control subject suffering from said.
 17. A kit ofclaim 15, wherein said agent is selected from a particle or fluorescentmarker-labeled anti-anucleated blood cell antibody, or wherein saidinstruction is selected from an instruction for bead-based anucleatedblood cells isolation, an instruction for FACS sorting of anucleatedblood cells, an instruction for anucleated blood cell recovery bycentrifugation, or negative selection of non-anucleated blood cellcomponents.
 18. A device for diagnosing cancer, the device comprising asupport and at least one agent for specifically determining a leveland/or activity of at least one nucleic acid mutant in an anucleatedblood cell sample of the subject attached to said support, and acomputer-readable medium having computer-executable instructions forperforming the method recited in claim
 1. 19. The device of claim 18,wherein said at least one agent is an oligonucleotide probe orsequencing primer.
 20. The device of claim 18, comprising a lateral flowdevice, a dipstick or a cartridge for performing a nucleic acidhybridization reaction between: an anucleated blood cells-extractednucleic acid and at least one nucleic acid mutation-specificamplification primer or oligonucleotide probe, wherein said nucleic acidmutation-specific amplification primer or oligonucleotide probe isspecific for a cancer-specific mutation, or an anucleated bloodcells-extracted nucleic acid and a plurality of gene-specificamplification primers or oligonucleotide probes for providing acancer-specific gene expression profile.
 21. The method of claim 10,wherein said nucleic acid is messenger ribonucleic acid (mRNA).